Physicochemical, Aroma Compounds, Microbial Community, and Antioxidant Capacity of Huangjiu-Based Functional Liqueur Fermented with Edible Herbs

Abstract

A functional Huangjiu-based liqueur (called by Lujiu in China), a type of Chinese rice wine, was developed by incorporating Chinese gall leaven, as a medicinal–edible homologous ingredient, into the fermentation process to enhance its bioactivity. The physicochemical properties and enzymatic activities were investigated and found that supplementation with 2% (v/v) Chinese gall leaven optimized fermentation efficiency and substrate utilization. The co-fermentation significantly elevated the concentrations of bioactive compounds and improved antioxidant capacity, particularly free radical scavenging activity. Compared to traditional Chinese rice wine, the supplemented variant exhibited markedly higher levels of malic acid and phenolic acids. GC-MS analysis identified 85 and 84 volatile flavor compounds in the two supplemented variants, respectively, exceeding the 70 compounds detected in traditional Huangjiu. GC-IMS further revealed significant enrichment of key alcohols (e.g., 3-methyl-1-butanol, 2-methyl-1-propanol) and aldehydes (e.g., propanal, acetaldehyde) in the supplemented group. Microbial community analysis indicated distinct shifts, with increased relative abundances of Pediococcus, Lactiplantibacillus, Aspergillus, and Saccharomyces in the Chinese gall leaven-supplemented fermentation. These results suggest that the native microflora and enzymatic systems of Chinese gall leaven could enhance microbial metabolism and fermentation efficiency, thus contributing to the unique characteristics of rice wine and providing a novel strategy for functional Huangjiu-based liqueur production.

1. Introduction

Huangjiu, also known as Chinese rice wine or yellow rice wine, is one of the oldest fermented alcoholic beverages in China, with a documented history of over 2500 years, alongside beer and grape wine. It is also considered one of China’s national alcoholic beverages [1]. Huangjiu has gained popularity among consumers due to its unique flavor, low alcohol content, and richness in bioactive compounds such as polyphenols, melanoidins, peptides, amino acids, and γ-aminobutyric acid, vitamins, and oligosaccharides [2]. Based on updated data from the National Bureau of Statistics of China and industry reports, Huangjiu production and market dynamics have evolved significantly in recent years. In 2019, Huangjiu production reached 3.53 million kiloliters (up from 2.5 million kiloliters in 2017), solidifying China’s global leadership in production and exports. By 2023, the sector demonstrated resilience despite structural adjustments: total production stood at 1.90 million kiloliters [1,3].

The brewing of Huangjiu involves a complex process including soaking, steaming, cooling, mixing with Qu, and fermentation, conducted in an open, multi-strain fermentation system that supports a diverse microbial community, ultimately contributing to its characteristic aroma, flavor, and color [1,4]. Consequently, microbial metabolism plays a crucial role in shaping the flavor and aroma of Huangjiu. Research indicates that Saccharomyces cerevisiae contributes to the production of alcohol and flavor compounds, while bacteria such as Lactobacillus, Weissella, and Pediococcus enhanced the formation of higher alcohols, volatile acids, and esters. Molds like Rhizopus played a role in the production of organic acids, and species such as Pediococcus pentosaceus and Rhizomucor emersonii were associated with aldehyde microbial metabolism [3,5]. Furthermore, it has been discovered that the co-fermentation of edible herbs and glutinous rice not only reduced the bitter taste while highlighting the rice wine with fruity herbaceous notes, resulting in a superior taste profile, but also brought about positive effects on fermentation efficiency and an exquisite flavor [6]. Hence, it would be a practicable strategy to ferment glutinous rice with edible herbs to develop novel Huangjiu-based liqueur.

Lujiu, as a category of Chinese functional wine, is also known as a health-care liqueur with a long history, which is highly favorable to Chinese consumers due to their perceived nourishing and tonic properties [7]. Huangjiu-based liqueur is made by combining the traditional alcoholic beverage of Chinese rice wine with functional compounds or ingredients [7]. With the rapid development of the health industry and the fast-growing demand for healthy diets and improved quality of life in the consumer market, the definition and status of Huangjiu-based liqueur has changed [5]. The China Alcoholic Drinks Association defined Lujiu as a specific beverage made with Huangjiu or Baijiu as the wine base, adding traditional medicine food homology substances as ingredients, involving technologies such as extraction, redistillation, or directly adding specific ingredients extracted from food. However, research on Huangjiu-based liqueur has long lacked systematic theoretical support, although interest in this area has been increasing in recent years.

In recent years, growing health awareness among consumers has led to increased demand for low-alcohol beverages with enhanced nutritional and health-promoting properties [8]. Conventional Huangjiu generally contains between 14 and 20% v/v of alcohol. According to the Chinese National Standard for Huangjiu (GB/T 13662-2018), products with an alcohol content not exceeding 12.0% v/v may be classified as low-alcohol Huangjiu [9]. In addition, Liu et al. noted that Huangjiu features low grain consumption, low alcohol content, and high nutrition, which conforms to the global trend of beverage alcohol consumption [10]. However, most of Lujiu prepared from Baijiu has the characteristics of high alcohol. In response, researchers have focused on developing Huangjiu-based liqueur via co-fermentation during the traditional Huangjiu brewing process. This approach incorporates medicinal, herbs, and fruits through co-fermentation, aiming to enrich bioactive substances such as phenolics and flavonoids, enhance antioxidant capacity, and optimize the composition of flavor esters to improve taste. Therefore, incorporating food homology herbs during the rice fermentation, aiming to improve both the nutritional value and functional properties, has been widely utilized [11,12].

Among the numerous food homology ingredients available in traditional Chinese herbs, Chinese gall leaven stands out due to its unique fermentation profile and rich content of bioactive compounds, including gallotannins, organic acids, and polyphenols, which are known for their strong antioxidant and antimicrobial properties [13]. Chinese gall leaven is a naturally fermented substance derived from gallnut powder inoculated with beneficial microorganisms, and it has historically been used for gastrointestinal health and detoxification in traditional medicine [14]. Importantly, Chinese gall leaven can interact with the microbial ecosystem and various molds involved in traditional rice wine during co-fermentation [15]. Furthermore, the integration of Chinese gall leaven into rice wine fermentation may significantly impact the flavor landscape by introducing new volatile compounds and enhancing the diversity of aroma components through its enzymatic activities and microbial metabolism [16,17]. Therefore, this study aimed to comprehensively characterize a novel low-alcohol Huangjiu-based liqueur enriched with bioactive constituents and exhibiting antioxidant activity, which was produced through co-fermentation with Chinese gall leaven, while the positive effect on physicochemical properties, aroma compounds composition, and microbial community structure were analyzed. This work provides theoretical support for advancing the study of how co-fermentation influences the active constituents of edible herbs and offers a basis for promoting the broader application of Huangjiu-based liqueurs.

2. Materials and Methods

2.1. Materials and Reagents

Glutinous rice was purchased from a local supermarket in Shaoxing, Zhejiang Province, China. Chinese gall leavens were obtained from Tongrentang Chinese Medicine Co., Ltd. (Beijing, China), labeled as CGL-1, and from Huqingyutang Pharmaceutical Co., Ltd. (Hangzhou, China), labeled as CGL-2. Wheat Qu and Jiuyao were supplied by Zhejiang Guyuelongshan Shaoxing Liquor Co., Ltd. (Shaoxing, China).

The 2,2-diphenyl-1-picrylhydrazyl (DPPH),2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Folin–Ciocalteu reagent and standards such as gallic acid, protocatechuic acid, ferulic acid, and various organic acids were obtained from Yuan-ye Biotech Co., Ltd. (Shanghai, China). All other chemical reagents were analytical grade and were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of Huangjiu-Based Liqueurs

The fermentation process of the Huangjiu-based liqueur (Lujiu) samples was conducted and optimized according to a previous study [18]. Briefly, glutinous rice was soaked in water at a ratio of 1:5 (m/v) at room temperature for 12 h, then steamed for 20 min. After cooling to room temperature, 10% wheat Qu and Jiuyao were mixed into the cooled glutinous rice along with 2% Chinese gall leaven powder. The mixture was then transferred into a traditional wide-mouth ceramic jar (with a material-to-vessel volume ratio of approximately 2:3). The jar was covered with a double layer of sterile gauze to prevent contamination while permitting minimal gas exchange, simulating a traditional semi-anaerobic fermentation environment. The fermentation was conducted at 30 °C for 5 days. The sample fermented with Tongrentang Chinese gall leavens (CGL-1) was labeled as CRW-CGL-1, while the sample with Huqingyutang Chinese gall leavens (CGL-2) was labeled as CRW-CGL-2, and the sample fermented without Chinese gall leaven powder was used as the control and labeled as CRW.

2.3. Physicochemical Analysis and Enzyme Activity Determination

The reducing sugar and total acid contents were determined according to a previous study [18]. The other physicochemical properties were analyzed following the method described by Yang et al. [19]. The alcohol content was measured according to the People’s Republic of China National Standard for fermented glutinous rice (GB/T 13662-2018, Huangjiu. Standards press of China: Beijing, China, 2018), and total amino acid nitrogen content was determined using the titratable acidity method. The pH values were measured using a digital pH meter (Schott Lab 850, Mainz, Germany), while soluble solid content was expressed as °Brix and measured with a handheld refractometer (Shanghai Lichen Instrument Technology Co., Ltd., Shanghai, China).

To estimate the activities of key enzymes, crude extracts were prepared as follows: 10 g of sample was mixed with 90 mL of deionized water and stirred for 20 min at room temperature. The supernatant was collected after centrifugation at 5000× g for 10 min at 4 °C. The amylase activity was measured based on the method described by Xu et al. [20], where one unit (U) of activity is defined as the amount of enzyme required to produce 1 μmol of reducing sugar from starch hydrolysis per minute. The glycase activity was assessed based on the definition that one unit (U) corresponds to the amount of enzyme required to hydrolyze 1 μmol of starch per minute. The protease activity was measured in an acidic buffer solution (pH 3.0). Briefly, 1 mL of buffer, crude enzyme extract, and casein were mixed and incubated for 10 min, followed by the addition of trichloroacetic acid to stop the reaction. The substrate consumption was quantified using the Lowry method with Folin–Ciocalteu reagent. The xylanase activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar from 5 mg/mL of xylan in a pH 5.5 buffer at 37 °C per minute. The β-glucosidase activity was determined and calculated based on our previous study [20].

2.4. Color, Turbidity, and Conductivity Determination

The color parameters of rice wine were measured using the CIELab method [21], including L* (lightness), a* (redness), and b* (yellowness) values. The variation in electric conductivity was assessed using a conductivity meter (Shanghai Yueping DDS-307, Shanghai, China) by placing the rice wine at 25 °C under continuous stirring for 5 min. Results were expressed in μS/cm to indicate the stability level (Δx), with values < 40 considered very stable and >80 indicating low stability [21].

The turbidity was determined by allowing the rice wine to stand at room temperature for 1 min before measurement using a portable turbidity meter (Shanghai Yuefeng SGZ-200BS, Shanghai, China). Results were reported in Nephelometric Turbidity Units (NTUs) after standard correction.

2.5. Assessment of Antioxidant Activities

The antioxidant properties of Huangjiu-based liqueur (Lujiu) were evaluated in vitro using spectrophotometric methods, based on changes in the absorbance of colored radical cations, as described in our previous studies [19,22]. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was assessed by mixing 1 mL of the sample with 4 mL of 0.1 mM DPPH solution and incubating in the dark for 90 min. Absorbance was then measured at 517 nm.

The ABTS radical cation was prepared by reacting 7 mM ABTS with 2.45 mM potassium persulfate (K₂S₂O₈) in the dark for 16 h. A 200 µL aliquot of sample or positive control was added to 800 µL of ABTS·⁺ solution, incubated at room temperature for 6 min, and absorbance was measured at 734 nm.

Hydroxyl radical scavenging activity was determined by mixing equal volumes of 0.75 mM FeSO₄, 0.75 mM 1,10-phenanthroline, 0.01% H₂O₂, and 0.15 M sodium phosphate buffer (pH 7.4). The mixture was incubated at 37 °C for 30 min before measuring absorbance at 536 nm. The radical scavenging capacity was expressed as a percentage of inhibition.

Ferric reducing antioxidant power (FRAP) was measured by mixing 25 µL of sample with 250 µL of FRAP working solution, composed of 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (in 400 mM HCl), and 20 mM FeCl₃ in a 10:1:1 (v/v/v) ratio. After incubation at 37 °C for 20 min, absorbance was measured at 593 nm. Results were expressed as micromoles of Fe (II) equivalents, based on an 8-point calibration curve of FeSO₄ (0.2–2.0 mM).

Total reducing power was determined by mixing 0.5 mL of sample with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. After incubation at 50 °C for 20 min, 2.5 mL of 10% trichloroacetic acid was added to terminate the reaction. The mixture was centrifuged, and the supernatant was combined with 0.5 mL of 0.1% ferric chloride solution. Absorbance was measured at 700 nm, with higher absorbance indicating greater reducing power.

2.6. Determination of the Content of Bioactive Constituent

The Folin–Ciocalteu method was used to measure the total polyphenol content following the procedure described by Xu et al. [20]. Gallic acid was used as the standard, and results were expressed as micrograms of gallic acid equivalents per milliliter of sample (μg GAE/mL) [23].

The total flavonoid content was measured using a colorimetric method. Briefly, 40 μL of sample and 200 μL of buffer were mixed with 20 μL of 5% sodium nitrite solution and incubated for 6 min. Then, 40 μL of 10% aluminum chloride solution was added and allowed to react for 5 min. Finally, 100 μL of 1 M sodium hydroxide was added, and the absorbance of the supernatant was measured at 510 nm. Results were expressed as micrograms of catechin equivalents per milliliter of sample (μg EC/mL).

The total saponin content was determined using the method described by Xu et al. [20]. In brief, 40 μL of the sample was mixed with 180 μL of 4% vanillin ethanol solution and 180 μL of 5 M sulfuric acid, followed by incubation in the dark for 20 min. The absorbance was then measured at 505 nm, and the results were expressed as micrograms of catechin equivalents per milliliter of sample (μg EC/mL).

2.7. Quantitative Analysis of Organic Acids and Phenolic Acids

The organic acids and phenolic acids were determined using a high-performance liquid chromatography (HPLC) system (LC-20A, Shimadzu, Kyoto, Japan) equipped with a reverse-phase C18 column (4.6 × 250 mm, 5 μm), following the method described in our previous study [24]. For phenolic acid analysis, the mobile phase consisted of deionized water (solvent A) and methanol containing 0.1% formic acid (solvent B). The gradient elution was programmed as follows: 5% B for 4 min, 5–10% B over 4 min, 10–20% B over 12 min, 20–33% B over 16 min, 33–50% B over 10 min, 50–75% B over 10 min, and 75–100% B for 5 min. Phenolic acids were detected at 280 nm and quantified based on the integrated peak areas using standard calibration curves.

As for organic acid analysis, an aqueous acetonitrile solution (3:97, v/v) containing 0.02 mol/L KH₂PO₄ was used as the mobile phase. The flow rate was set at 0.8 mL/min, and detection was performed at 210 nm. The concentrations of identified compounds were determined using calibration curves of authentic standards and expressed as micrograms per milliliter of Huangjiu-based liqueur.

2.8. GC-IMS Analysis

The volatile compounds in Huangjiu-based liqueur samples were analyzed using a gas chromatography–ion mobility spectrometry (GC-IMS) instrument (Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany) with headspace sampling, following the method of Yu et al. with minor modifications [25]. Each sample (2.0 g) was placed in a 20 mL headspace vial and incubated at 80 °C for 30 min. Afterwards, the headspace gas was then injected into a WAX-CB-1 capillary column (30 m × 0.53 mm) using heated syringes maintained at 85 °C. High-purity nitrogen was used as the drift gas at a flow rate of 150 mL/min. The temperature program was as follows: the column was held at 60 °C, with an initial flow rate of 2 mL/min for 2 min, gradually increased to 100 mL/min over the next 18 min, and maintained at 100 mL/min for 45 min. Ketones (C4–C9) were used as external standards to calculate the retention indices of the volatile compounds. All analyses were conducted in triplicate, compound identification was based on drift times and comparison with standards in the GC-IMS database. The Reporter plug-in was used to compare the spectrograms among samples, including 3D spectrograms, 2D top views, and differential spectrograms. The Gallery Plot plug-in was employed to compare the relative contents of identified volatile compounds.

2.9. HS-SPME-GC-MS Analysis

The identification of volatile aroma compounds was performed as previously described with slight modifications [26]. Briefly, 5 g of wine sample and 20 μL of internal standard (2-octanol, 2.0 mg/L) were placed into a 20 mL headspace bottle. The volatile compounds were extracted using a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber (Supelco, Inc., Bellefonte, PA, USA) at 60 °C for 40 min. Extracted volatiles were then desorbed in the GC injection port at 250 °C for 5 min. The analysis was carried out on a GC-MS system (SH-Rxi-5Sil MS; Shimadzu Corporation, Kyoto, Japan) equipped with a DB-5MS column (30 m × 0.25 mm, 0.25 μm). Helium was used as the carrier gas at a flow rate of 2 mL/min. The column chamber was initially held at 40 °C for 3 min, then increased at 5 °C/min to 120 °C and held for 2 min, followed by an increase to 200 °C at 8 °C/min and held for another 2 min. The quality detector was operated in electron shock mode at 70 eV, with the ion source temperature set to 230 °C. The volatile organic compounds were identified by comparing mass spectra against libraries (NIST 17.0) and retention indices (Kovats Indices, KIs) from the literature, then were quantified via the internal standard method. The contribution of these important odor-active compounds to the overall aroma was evaluated using their odor activity values (OAVs). The OAVs were calculated by dividing the concentration of each odor-active compound by its corresponding odor threshold [7].

2.10. Sensory Evaluation

The method of sensory evaluation was performed as previously described with slight modifications [27]. Sensory evaluation of Huangjiu-based liqueur was conducted on CRW, CRW-CGL-1, and CRW-CGL-2, each of which was heated in an 80 °C water bath for 5 min. The samples were evaluated based on six sensory descriptors: clarity, bouquet, herbaceous note, fullness, coordination, and color/luster. The analysis was performed by a trained panel consisting of 12 members (6 females and 6 males), aged between 20 and 30 years, who have received training in standard taste solutions. Samples were served in transparent glasses at a temperature of 40 °C and presented in randomized order to avoid bias. Members of the sensory group sipped each sample (10 mL), held it in mouth for 20 s and then spit it out. After each evaluation, the taster rinsed the mouth with pure water and rested for 3 min to relieve fatigue. The evaluation is repeated three times. Four grades, excellent, good, middle, and bad, were set to rate and divide, using a 16-point interval scale: 0–4 (marginally present), 5–8 (clearly present), 9–12 (highly pronounced), and 13–16 (overwhelmingly pronounced). The average scores for each descriptor were calculated and analyzed using one-way analysis of variance (ANOVA) in SPSS 25.0.

2.11. High-Throughput Sequencing

According to the previous study, total DNA was extracted following the instructions of the FastDNA SPIN Kit (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China) [24]. The DNA concentration and purity were assessed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and DNA quality was evaluated by 1% agarose gel electrophoresis. The fungal ITS1–ITS2 region and the bacterial V3–V4 region of the 16S rDNA genes were amplified using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′)/ITS2R, (5′-GCTGCGTTCTTCATCGATGC-3′), and 338F (5′-ACTCCTACGGGAGGCAGCAG-3′)/806R (5′-GGACTACHVGGGTWTCTAAT-3′), respectively. The resulting PCR products were purified and used to construct a PCR amplicon library for high-throughput sequencing on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA). The alpha diversity analysis was performed to evaluate the diversity and richness of microbial communities, with indices including Chao, ACE, Shannon, and coverage index used to estimate species richness, diversity, and sequencing coverage. The clustered OTUs results were homogenized, and Venn diagrams were created.

2.12. Statistical Analysis

The results were expressed as mean ± standard deviation, based on experiments conducted in triplicate. The statistical significance was determined at a p-value < 0.05. Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test, performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA) and Origin 2021 (OriginLab Corporation, Northampton, MA, USA). The qualitative identification of volatile compounds was carried out using the NIST and IMS databases and visualized with the Reporter and Gallery Plot plug-ins. Principal component analysis (PCA) was performed using SIMCA software (version 14.1.0.2047).

3. Results and Discussion

3.1. Effect of Co-Fermentation on the Physicochemical Characteristics of Huangjiu-Based Liqueur

The different Huangjiu-based liqueur (Lujiu) fermentation samples were obtained, and their physicochemical properties are recorded. Bilateral microbial fermentation and metabolism are known to produce various organic acids, leading to a decrease in pH and an increase in total acid content [14]. During the fermentation process of Huangjiu, various complex biochemical reactions occur, including the Maillard reaction between carbonyl groups and amino groups. These reactions not only contribute to the unique and desirable sensory characteristics of Huangjiu but also cause dynamic fluctuations in physicochemical properties [28].

A rapid decline in pH was observed during the first 48 h (Figure 1 and Figure S1). As a crucial environmental factor that directly affects the succession of microbial populations, the content of total acid exhibited an increased tendency, with the highest recorded value (4.27 g/kg) occurring in the Chinese rice wine fermented with Huqingyutang Chinese gall leaven fermented (CGL-CRW-2). Amino acid nitrogen is a key indicator reflecting the overall level of amino acids and small peptides of Huangjiu. Compared to CRW, both CRW-CGL-1 and CRW-CGL-2 had higher amino acid nitrogen levels. The alcohol content in Huangjiu typically ranges from 8% to 18%, and the alcohol levels of all three samples fell within this range. Meanwhile, the contents of reducing sugars and total solids exhibited a decreasing trend, whereas the value of soluble solids increased during the first 48 h and subsequently declined. Wang et al. investigated the volatile compounds in eight broomcorn millet Huangjiu samples collected from different brewing stages using solvent-assisted evaporative extraction combined with GC-MS and chemometric analysis. Their findings revealed that most volatile compounds were formed during the primary fermentation stage, and the aging time significantly influenced the composition and proportion of these volatile components [5].

Figure 1.

Physicochemical properties analysis at the endpoint of CRW-CGL and CRW fermentation (120 h), including pH value, total acid, amino acid nitrogen, ethanol content, reducing sugar content, total solids, and soluble solids. (a–c: Different letters in peer data indicate significant differences between groups (p < 0.05)).

The color analysis showed that both L* and b* values increased after the addition of Chinese gall leave (CGL), indicating that the color of CGL-CRW became brighter and more yellow compared to CRW (Figure 2A). This can be attributed to the CGL promoting the extraction of more pigments from the raw materials into the Huangjiu. Additionally, previous studies have suggested that anthocyanidins may influence beverage color and are positively correlated with the b* value [28]. Furthermore, it was observed that the turbidity of CGL-CRW-1 and CGL-CRW-2 was lower than that of CRW, suggesting improved the clarity of wine. Electrical conductivity is an important indicator for evaluating the stability of wine [29]. The various substances in Huangjiu, including acids, organic acid salts, mineral salts, and other compounds, exist as charged ions and form an electrolyte solution during the fermentation process [30,31]. Among the samples, CGL-CRW-1 exhibited the highest electrical conductivity (846.66 ± 0.47 μS/cm), followed by CGL-CRW-2 (800.58 ± 4.50 μS/cm) and CRW (788.66 ± 1.20 μS/cm) (Figure 2B).

Figure 2.

Phytochemical properties and microbial abundance of CRW-CGL and CRW. (A) L, a, b values; (B) electrical conductivity and turbidity; heatmap analysis of the abundant fungal (C) and bacterial (D) genera present in CGL-CRW and CRW. The color intensity was proportional to the relative abundance of bacterial/fungal genera. * indicate significant differences between groups (p < 0.05).

3.2. Effect of Co-Fermentation on the Enzyme’s Activities During Huangjiu-Based Liqueur Fermented Process

The fermentation process of Huangjiu requires the synergistic action of glycosylase, α-amylase, and protease. Glycosylase and α-amylase continuously convert starch into reducing sugars, which not only support microbial growth and proliferation but are also eventually converted into alcohol during the later stages of fermentation [32,33]. The acid protease plays a critical role in wine fermentation by effectively breaking down proteins into amino acids and peptides, thereby providing essential nutrients and aroma precursors [34].

As shown in Figure 3 and Figure S2, the enzyme activity trends across different samples were generally similar, though their activity levels varied at different fermentation stages. The amylase activity peaked at 96 h, the trend in saccharifying enzyme activity was comparable to that of amylase. The activity in CGL-CRW and CRW peaked at 48 h and 72 h, respectively. Throughout the fermentation process, CGL-CRW-2 exhibited the highest enzymatic activity. Both β-glucosidase and xylanase contributed to breaking down the grain cell wall, promoting the release of intracellular starch and proteins, and converting polysaccharides in the cell wall into fermentable sugars. This improved the efficiency of raw material utilization. At the post-fermentation stage, β-glucosidase activity was higher in CGL-CRW than in CRW, similar with xylanase. In sum, in comparison to CRW, the enhanced enzymatic activities observed in CGL-CRW, particularly the earlier peaks in saccharifying enzymes and sustained β-glucosidase/xylanase activity during post-fermentation, align with previous findings that complex microbial consortia (such as those in Chinese gall leaven) can modulate enzyme secretion dynamics to optimize substrate degradation [34]. This suggests that the unique microbial assemblage in CGL-CRW not only accelerates starch-to-sugar conversion but also strengthens cell wall breakdown, a dual advantage that likely contributes to its superior raw material utilization efficiency. Conversely, the delayed or lower enzymatic activity in CRW may reflect a less synergistic microbial–enzyme interaction, limiting its capacity to hydrolyze complex polysaccharides and proteins within the same timeframe.

Figure 3.

Crucial enzymes activities at the endpoint of CRW-CGL and CRW fermentation (120 h), including amylase activity, saccharifying enzyme activity, acid protease activity, β-glucosidase enzyme activity, and Xylanase activity. (a–c: Different letters in peer data indicate significant differences between groups (p < 0.05)).

Notably, the higher acid protease activity in CGL-CRW-2 at 24 h. Between 24 h and 72 h, the activity of CGL-CRW-1 and CGL-CRW-2 decreased sharply, contrasting with the more stable but lower activity in CRW, implying that Chinese gall leaven-derived microorganisms may trigger an earlier burst of proteolytic activity, potentially enriching the fermentation matrix with amino acids and aroma precursors earlier in the process, which could positively influence the flavor complexity of the final Huangjiu [5]. These differences highlight the critical role of microbial diversity in driving enzymatic synergy, offering a mechanistic basis for why Chinese gall leaven are valued for improving both fermentation efficiency and product quality.

3.3. Effect of Co-Fermentation on the Phenolic Bioactive Compounds, Organic Acids, Phenolic Acids, and Antioxidative Capabilities of Huangjiu-Based Liqueur

3.3.1. Phenolic Bioactive Compounds of Huangjiu-Based Liqueur

Phenolics, flavonoids, and saponins are naturally occurring bioactive compounds in Chinese gall leavens. In rice wine, these broad-spectrum functional components play a key role in maintaining redox homeostasis and serve as critical indicators of nutritional quality [17]. To quantify these bioactive compounds, high-performance liquid chromatography was employed to determine the total contents of phenolics, flavonoids, and saponins.

As shown in Table 1, the total phenolic content was highest in the ethanol extract of CGL-CRW-2 (247.93 μg/mL) and lowest in the ethanol extract of CRW (39.45 μg/mL). Compared with the CRW extract, the total phenolic content in the ethanol extracts of CGL-CRW increased by 5.6-fold and 6.3-fold, respectively. The nutritional composition of rice is characterized by its high starch and B vitamin content, while exhibiting relatively low levels of phenolics and flavonoids [35]. Microorganisms involved in fermentation can produce or release phenolic compounds either through secondary metabolic pathways or through the action of extracellular enzymes. Moreover, the CRW is primarily metabolized and synthesized by non-yeast microorganisms to enhance the total phenolic content. The difference in total phenolic content between CGL-CRW and CRW suggests that the addition of Chinese gall leaven contributed substantially to the enhancement of phenolic content in rice wine, which is consistent with reports identifying these as major bioactive components in raw Chinese gall leaven extracts. This confirms that co-fermentation effectively transfers these functional compounds into the beverage. As a fermented traditional Chinese medicine, CGL provides microbial hydrolytic enzymes during fermentation that break down the cellulose backbone and phenolic branches, thereby increasing the levels of free phenolics and flavonoids [18]. The results in total flavonoids and saponins were like those of total phenolic content, indicating that using Chinese gall leaven as a fermentation ingredient promoted the enrichment of phenolics, flavonoids, and saponins substances in rice wine. Notably, CRW-CGL-2 consistently yielded the highest levels of total phenolics, flavonoids, and saponins among all samples, indicating that CGL-2 may be particularly effective in enriching these bioactive compounds.

Table 1.

Phenolic acids analysis in CGL-CRW and CRW.

Phenolic Acids (mg/mL)	CGL-CRW-1		CGL-CRW-2		CRW
	E-1	W-1	E-2	W-2	E-3	W-3
Gallic acid	186.61 ± 2.24 Ab,*	144.53 ± 2.33 Bd	203.99 ± 1.79 Aa	166.15 ± 3.74 Bc	1.20 ± 0.03 Bf	11.31 ± 0.13 Ae
Gallocatechin	2.64 ± 0.04 Be	15.73 ± 0.24 Aa	5.06 ± 0.04 Bc	7.96 ± 0.07 Ab	4.05 ± 0.05 d	ND
Protocatechuic acid	3.90 ± 0.03 Ab	2.97 ± 0.07 Bd	4.61 ± 0.04 Aa	3.71 ± 0.04 Bc	0.44 ± 0.03 Bf	0.94 ± 0.02 Ae
Catechin	1.61 ± 0.05 d	ND	9.52 ± 0.06 Aa	2.87 ± 0.04 Bc	5.13 ± 0.06 b	ND
Epigallocatechin	ND	3.92 ± 0.05 c	ND	1.72 ± 0.03 d	5.28 ± 0.05 Aa	5.17 ± 0.06 Ab
Epigallocatechin gallate	0.94 ± 0.01 Be	1.45 ± 0.08 Ac	3.18 ± 0.03 Aa	0.57 ± 0.02 Bf	1.06 ± 0.04 Bd	2.21 ± 0.05 Ab
Caffeic acid	1.32 ± 0.02 Aa	0.81 ± 0.04 Bc	0.51 ± 0.02 Be	1.14 ± 0.05 Ab	0.62 ± 0.04 Bd	1.19 ± 0.04 Ab
Epigallocatechin gallate	1.03 ± 0.04 c	ND	0.33 ± 0.02 Bd	1.40 ± 0.03 Ab	1.04 ± 0.06 Bc	1.92 ± 0.05 Aa
Epicatechin	3.23 ± 0.03 Be	4.76 ± 0.07 Ac	0.58 ± 0.04 Bf	5.32 ± 0.02 Ab	3.59 ± 0.07 Bd	6.73 ± 0.04 Aa
Vanillin	0.63 ± 0.02 Ac	0.75 ± 0.05 Ac	0.40 ± 0.01 Bd	0.83 ± 0.03 Ab	0.70 ± 0.04 Bc	1.13 ± 0.06 Aa
Cinnamic acid	5.33 ± 0.03 Aa	1.47 ± 0.02 d	4.65 ± 0.03 Ab	1.70 ± 0.02 Bc	1.71 ± 0.05 Ac	1.72 ± 0.05 Ac
Guaiacol	5.39 ± 0.07 Bc	9.41 ± 0.07 Aa	3.15 ± 0.04 Be	5.45 ± 0.07 Ac	7.74 ± 0.07 Ab	4.64 ± 0.05 Bd
Ferulic acid	6.35 ± 0.04 Ab	3.66 ± 0.06 Be	7.21 ± 0.04 Aa	4.55 ± 0.05 Bd	4.88 ± 0.03 Ac	3.11 ± 0.05 Bf
Vanillin	ND	0.68 ± 0.06 b	ND	ND	ND	1.10 ± 0.02 a
Quercetin	3.28 ± 0.04 Aa	1.71 ± 0.04 Be	3.15 ± 0.03 Ab	1.56 ± 0.04 Bf	2.02 ± 0.06 Bd	2.39 ± 0.02 Ac
Kaempferol	0.10 ± 0.01 b	ND	1.59 ± 0.03 a	ND	ND	ND
Vanillic acid	ND	ND	0.14 ± 0.01	ND	ND	ND
Total content	222.36 ± 2.72 b	191.85 ± 3.18 d	247.93 ± 2.23 a	204.93 ± 4.25 c	39.45 ± 0.68 f	43.55 ± 0.64 e

* The sample was concentrated through the process of evaporation and subsequently subjected to freeze-drying. The freeze-dried powder was extracted from ethanol and water respectively.”E-1” and “E-2” represented the ethanol extract of CGL-CRW-1 and CGL-CRW-2, respectively. “E-3” represented the ethanol extract of CRW. The water extract was named “W-1”, “W-2”, and “W-3”. Data are shown as mean ± standard deviation of three sets of replicated trials; each set of data was analyzed via one-way ANOVA to mark significant differences (p < 0.05). ND means that the substance was not detected. For each compound, different lowercase letters (a–f) indicate significant differences (p < 0.05) among the three wine samples across the row. For each sample (column), different uppercase letters (A, B) indicate significant differences among the different extraction methods (e.g., ethanol vs. water extract) within that column.

3.3.2. Organic Acids and Phenolic Acids Content of Huangjiu-Based Liqueur

Organic acids contribute to the acidity of Chinese rice wine and play a critical role in inhibiting microbial growth and influencing the formation of esters. The types and contents of organic acids in CGL-CRW and CRW were determined, and the results are shown in Table 2. Significant differences (p < 0.05) were observed in the levels of lactic acid, malic acid, formic acid, tartaric acid, and oxalic acid between CGL-CRW and CRW. The contents of lactic acid, formic acid, tartaric acid, and oxalic acid were significantly higher in CRW than in CGL-CRW-1 and CGL-CRW-2 (p < 0.05), whereas malic acid content was significantly higher in the CGL-CRW samples. This suggests that the addition of CGL promoted an increase in malic acid content in Huangjiu. There were insignificant differences in organic acid contents between CGL-CRW-1 and CGL-CRW-2.

Table 2.

Qualitative and quantitative analysis of organic acids in CGL-CRW and CRW.

Organic Acids	CGL-CRW-1	CGLCRW-2	CRW
Oxalic acid	4.85 ± 0.07 ab,*	4.68 ± 0.17 b	5.13 ± 0.18 a
Tartaric acid	10.14 ± 0.28 b	10.20 ± 0.36 ab	10.88 ± 0.29 a
Formic acid	84.66 ± 2.32 b	78.71 ± 2.03 b	102.16 ± 7.31 a
Malic acid	26.77 ± 3.27 a	23.12 ± 3.05 a	10.19 ± 1.91 b
Malonic acid	57.02 ± 1.28 a	52.72 ± 2.77 a	56.85 ± 0.94 a
Lactic acid	39.97 ± 1.71 ab	35.77 ± 1.96 b	42.29 ± 3.27 a
Acetic acid	13.01 ± 0.32 a	13.04 ± 0.97 a	12.87 ± 0.67 a
Maleic acid	0.09 ± 0.01 a	0.09 ± 0.01 a	0.10 ± 0.02 a
Succinic acid	110.83 ± 3.81 a	117.71 ± 1.14 a	75.77 ± 2.83 a
Propionic acid	33.48 ± 1.84 a	19.15 ± 2.32 b	33.61 ± 2.19 a
Butyric Acid	774.38 ± 5.29 b	797.79 ± 7.52 a	648.83 ± 10.16 c

* Data are shown as mean ± standard deviation of three sets of replicated trials; each set of data was analyzed by one-way ANOVA to mark significant differences (p < 0.05). For each organic acid (row), different lowercase superscript letters (a, b, c) indicate statistically significant differences (p < 0.05) in its content among the three wine samples (CRW, CRW-CGL-1, CRW-CGL-2) as determined by Duncan’s multiple range test.

The phenolic acids in Huangjiu primarily originate from the raw brewing materials and the action of microbial fermentation, particularly through the production of feruloyl esterase [36]. Following the addition of CGL, the total phenolic acid content in Huangjiu increased significantly (p = 0.015). Modern pharmacological studies indicate that the main active components in CGL are tannins and gallic acid, and fermentation enhances gallic acid levels, which may explain this increase [12]. Furthermore, cell walls in wheat koji and rice were degraded by highly active ferulate esterase, xylanase, and cellulase metabolized produced by the microorganisms present in Chinese gall leaven. This enzymatic degradation facilitated the release of phenolic acids, thereby increasing their concentration in CGL-enriched Huangjiu [37].

3.3.3. Antioxidative Properties of Huangjiu-Based Liqueur

Polyphenolic compounds have been confirmed to exert strong antioxidant activity. They function as antioxidants by interrupting chain oxidation reactions, donating hydrogen atoms, scavenging free radicals, or chelating metal ion [38]. To extract antioxidant compounds from CGL-CRW and CRW samples, water and ethanol (80% ethanol), as two solvents with different polarities and solubility profiles, were selected according to our previous studies [39]. As shown in Figure 4A, across the tested concentration range, the DPPH radical scavenging activity of all extracts increased with concentration. The scavenging rate became moderate when concentrations exceeded 2.5 mg/mL. At the highest tested concentration, the DPPH radical scavenging rates were 97.2% (ethanol extract—CGL-CRW-1), 95.2% (ethanol extract—CGL-CRW-2), 63.2% (ethanol extract—CRW), 93.5% (water extract—CGL-CRW-1), 92.1% (water extract—CGL-CRW-2), and 55.0% (water extract—CRW). Overall, ethanol extract—CGL-CRW-1 exhibited the strongest DPPH scavenging activity, while the water extract of CRW showed the weakest. These results were similar to the ABTS radical scavenging activity results (Figure 4B). The hydroxyl radical (OH) scavenging activity also increased with extract concentration. For ethanol extracts, the scavenging rate increased from 45.4% to 76.8%, while for water extracts, it increased from 15.1% to 47.5% (Figure 4C). Bai et al. previously extracted a low molecular weight polysaccharide with higher dose-dependent DPPH·, ABTS⁺, and OH· scavenging activity. HP1 exhibited significant protection of HepG2 cells from H₂O₂ damage from a traditional Chinese rice wine, Guandong Hakka Huangjiu [28].

Figure 4.

The antioxidative activities and total contents analysis of bioactive compounds in CGL-CRW and CRW. (A) DPPH clearance rate. (B) ABTS clearance rate. (C) OH-clearance rate. (D) total reducing power. (E) FRAP. (F) The content of bioactive constituents. (E-1, E-2, and E-3 represented ethanol extracts of CGL-CRW-1, CGL-CRW-2, and CRW, respectively; W-1, W-2, and W-3 represented water extracts of CGL-CRW-1, CGL-CRW-2, and CRW, respectively).

Total reducing power and ferric reducing antioxidant power (FRAP) assays were also employed to evaluate antioxidant capacity. As shown in Figure 4D, the reducing power increased with extract concentration, with the highest value observed in ethanol extract CGL CRW 2 (0.666) at 20 mg/mL. The reducing powers of ethanol and water extracts of CRW were similar and significantly lower than those of the CGL-added samples. Likewise, FRAP values showed a similar trend, with ethanol extract CGL CRW 2 exhibiting the highest value (1155 μmol Fe²⁺/L), which was approximately eight times higher than the lowest value (138.7 μmol Fe²⁺/L) among all samples at the highest concentration tested (Figure 4E). In summary, rice wine fermented with Chinese gall leaven demonstrated significantly stronger free radical scavenging activity (against DPPH, ABTS, and OH), higher total reducing power, and greater ferric ion reducing capacity. These findings suggest its potential to alleviate excessive oxidative stress in vivo. Furthermore, ethanol extracts showed better antioxidant activity than water extracts in vitro.

3.4. Effect of Co-Fermentation on the Aroma Compounds of Huangjiu-Based Liqueur

HS-SPME-GC-MS was employed to detect the volatile compounds in different Huangjiu samples. A total of 86 volatile flavor substances, including 34 esters, 17 alcohols, 5 aldehydes, 4 acids, 5 ketones, and 21 other compounds, were identified across the three samples: 85 compounds in CGL CRW-1, 84 in CGL CRW-2, and 70 in CRW. Esters were the most diverse aroma compounds, while alcohols had the highest overall content. Among the samples, most aroma compounds were highest in CGL CRW-1, followed by CGL CRW-2 and CRW.

Ester compounds, which contribute plant and fruit like aromas, are mainly synthesized through microbial metabolism via acetyl CoA catalyzed by alcohol acyltransferase and are influenced by various fermentation factors [35]. Among the detected esters, ethyl palmitate had the highest concentration, followed by ethyl caprylate, ethyl caprate, ethyl hexanoate, ethyl elaidate, and ethyl myristate, which together accounted for 82.95% to 83.83% of total ester content. CGL CRW 1 had the highest concentration of ester compounds (18,766.9 ± 125.98 μg/g), while CGL CRW-2 exhibited the highest variety (34 types). Additionally, compared to CRW, 2-ethylhexyl acetate (soil and herbal aroma), isoamyl decanoate (rose aroma), and ethyl 10-undecenoate (fruity and fatty aroma) were uniquely present in both CGL CRW-1 and CGL CRW-2. These findings suggest that adding Chinese gall leaven to the fermentation process enhanced the ester profile of Huangjiu.

The primary pathways for alcohol production in fermented rice wines are sugar metabolism and dehydrogenation or decarboxylation of amino acids [40]. Among the detected alcohols, 3-methyl-1-butanol was the most abundant, followed by phenethyl alcohol, 1-octanol, 1-nonanol, and 2-ethylhexanol. These five accounted for 94.92%, 94.31%, and 95.81% of total alcohol content in CGL CRW 1, CGL CRW 2, and CRW, respectively. Notably, 1-heptanol (oily, citrus aroma) and lavandulol (lavender, spicy aroma) were only detected in the CGL CRW samples.

Aldehydes are generally formed via oxidation of polyphenols or higher alcohols under aerobic conditions or through amino acid degradation via the Strecker reaction [41,42]. Five aldehydes were identified, with phenylacetaldehyde showing the highest content. Apart from phenylacetaldehyde and stearaldehyde, other aldehydes were only found in the CGL-CRW samples, indicating that they likely originated from the Chinese gall leaven. Ketones, which often contribute unique aromas, were found in all three samples. These included 2-nonanone (milk, soap), 2-decanone (fatty), 4-isopropylcyclohex-2-en-1-one (coriander, woody), 2-undecanone (orange, green), and 2-hexadecanone (banana, pear). CGL-CRW-1 had the highest total ketone content (335.89 ± 3.22 μg/g). Other volatile compounds included terpenes, furans, alkanes, and their derivatives.

The higher alcohols and their corresponding acetate esters are critical determinants of the flavor and quality of rice wine [43]. Detected by GC-MS in Figure 5, the levels of 3-methyl-1-butanol (fusel), 1-hexanol (floral), and phenethyl alcohol (rose, fruity) were significantly higher in CGL-CRW-1 and CGL-CRW-2 than in CRW (p = 0.008), whereas 1-octanol (metallic) was higher in CRW. 1-nonanol (fatty, green) was lowest in CGL-CRW-2. These higher alcohols are mostly produced via sugar metabolism or through the Ehrlich pathway involving aromatic amino acids in S. cerevisiae [44]. Acetate esters are synthesized from acetyl-CoA and ethanol or higher alcohols during yeast metabolism, imparting floral and fruity notes to Huangjiu [8]. CRW exhibited the highest levels of isoamyl acetate and ethyl phenylacetate, followed by CGL-CRW-1 and CGL-CRW-2 (p = 0.041). Notably, 2-ethylhexyl acetate, associated with herbal and earthy aromas, was only detected in CGL-CRW-1 and CGL-CRW-2, contributing to their unique aromatic profile. The significant increase in acetate esters and higher alcohols in CGL-CRW is likely to contribute to a more intense fruity and floral aroma profile.

Figure 5.

Aroma heatmap analysis of CGL-CRW and CRW by GC-MS (A). Distribution of different kinds of volatile substances in samples (B). Qualitative and quantitative analysis of higher alcohols and ethyl ester compounds in samples (C). The radar map of CGL-CRW and CRW sensory evaluation (D).

3.5. Effect of Co-Fermentation on the Aroma Compounds Content in Huangjiu-Based Liqueur

A comparison plot illustrating the differences in volatile compounds among the samples is shown using the CRW-CGL-1 spectrum as the reference (Figure 5A). In the resulting plot, white indicates compounds present at the same concentration as the reference, red indicates a higher concentration relative to the reference, and blue indicates a lower concentration. The results showed that the types of volatile compounds in the samples were largely similar, while the signal intensities of individual substances varied.

To visually reveal and compare the relative contents of different volatile compounds in different samples, all signal peaks were extracted and used to construct a characteristic fingerprint (Figure 5B). In this figure, brighter colors represent stronger signals (higher concentrations), while darker colors indicate weaker signals (lower concentrations). Figure 5B shows that the volatile compound profile of Chinese rice wine changed notably after fermentation with Chinese gall leaven. In the red zone, most detected esters, such as ethyl octanoate, ethyl heptanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and (E)-ethyl-2-hexenoate, decreased. Conversely, in the yellow zone, the concentrations of several alcohols (3-methyl-1-butanol, 2-methyl-1-propanol, 1-hexanol, 3-methyl-1-pentanol, 1-butanol, linalool, and 4-terpinenol), aldehydes (propanal, acetaldehyde, acrolein), and a few esters (propyl acetate, ethyl propanoate, and methyl acetate) generally increased following the addition of Chinese gall leaven. Furthermore, Chinese gall leaven from different sources led to variations in the volatile compound profiles. Specifically, CRW-CGL-1 had relatively higher signal intensities for key aldehydes such as propanal and acetaldehyde, whereas CRW-CGL-2 showed stronger signals for several alcohol and ester compounds. These differences offer potential for diversifying the flavor profiles of Chinese gall leaven-fermented Huangjiu. Li et al. developed sequential inoculation millet Huangjiu with a harmonious aroma combined with moderate sweetness and acidity, fostering the development of diversified and customized Huangjiu [43].

The contribution of volatile compounds to the overall aroma of each wine sample was further assessed using odor activity values (OAVs) [45]. Generally, aroma compounds with OAVs greater than 1 are considered to significantly contribute to specific aroma characteristics of rice wines, while those with OAVs below 0.1 are regarded as having minimal aromatic impact [46]. A total of 42 aroma compounds had OAVs of more than 1, which were detected from all these three samples (Table S1). Isoamyl acetate, ethyl heptanoate, ethyl heptanoate, ethyl caprylate, phenethyl acetate, ethyl nonanoate, gamma-nonanolactone, ethyl caprate, palmitic acid ethyl ester, 1-Nonanol, 2-Nonanone, and 4-Hydroxy-3-methoxystyrene had higher OAVs in the three samples. Among them, isoamyl acetate ethyl heptanoate, ethyl heptanoate, ethyl caprylate, and 4-Hydroxy-3-methoxystyrene made significant contributions to the distinctive fruity and floral aroma characteristics of the Chinese rice wine. In addition, compounds such as isoamyl caprate, 4-ethyl-2-methoxyphenol, β-caryophyllene, undecanal, and 1-heptanol were detected exclusively in the CGL-CRW groups, thereby contributing to the overall aroma differentiation among the three groups’ products [31]. Notably, only capric acid isoamyl ester (rose) had OAV ≥ 1 in CGL-CRW-2. Aroma compounds including phenethyl acetate, ethyl lactate, 2,3-butanediol, 1-heptanol, nonane aldehyde, 4-hydroxy-3-methoxystyrene, and α-octyl cinnamyl alcohol had the highest OAVs in CGL-CRW-2, contributing notably to the floral and herbal aroma components of the bouquet, which was consistent with the sensory evaluation results [15]. In addition, palmitic acid ethyl ester, 3-Methyl-1-butanol, 1-Octen-3-ol, 1-Nonanol, 2-Nonanone, 2-Decanone, and 4-Ethyl-2-methoxyphenol exhibited higher OAVs in CGL-CRW-1, which contributed to the enhanced harmony of the wine. These findings indicated that there were significant aroma differences among the rice wines, potentially due to the influence of different components of raw materials on the microbial metabolic pathway, thereby causing changes in the bio-transformation of esters, as well as differences in the reactions between organic acids and higher alcohols [45].

3.6. Effect of Chinese Gall Leaven Fermentation on the Sensory Evaluation of Huangjiu-Based Liqueur

To describe the sensory differences among the samples fermented with different materials, quantitative descriptive analysis was conducted, and the sensory scores are presented in Figure 5D. A total of six aroma and taste attributes were assessed to develop the sensory profiles. CGL-CRW-2 exhibited the highest intensity of herbaceous and bouquet aromas among the three samples (p = 0.032), while color and luster showed insignificant difference between CGL-CRW-1 and CGL-CRW-2. However, CGL-CRW samples demonstrated significantly higher scores for color and luster, clarity, caramel-like, fullness, and bouquet compared to CRW (p = 0.022), whereas only the coordination attribute remained similar between CGL-CRW and CRW. The incorporation of Chinese gall leaven promoted the biosynthesis of key flavor compounds, notably acetate esters (e.g., isoamyl acetate and phenethyl acetate, which impart fruity notes), aromatic alcohols (such as phenethyl alcohol with its floral character), aldehydes and terpenes (contributing herbaceous nuances), as well as characteristic organic acids (e.g., malic acid). The synergistic enrichment and refinement of these compounds endowed the Huangjiu-based liqueur with more pronounced fruity, floral, and fresh herbaceous aromas, along with a better-balanced and fuller overall flavor profile [44].

The differences in aroma properties are mainly attributed to the characteristics of the CGL and the types and concentrations of volatile compounds produced during fermentation. The bouquet and herbaceous aromas are associated with the content of esters and higher alcohols, which contributed to the enhanced overall aroma of CGL-CRW [36]. Some esters (ethyl hexanoate, ethyl laurate) and aldehydes (undecanal, glycerol formal) can endow Huangjiu with fruity and citrus aromas, respectively, and phenylethanol has a sweet aroma similar to that of rose and honey. Additionally, the unique volatile compounds in CRW-CGL (cedrol, 4-Ethyl-2-methoxyphenol) note spicy and wood.

3.7. Effect of Chinese Gall Leaven Fermentation on the Microbial Diversity of Huangjiu-Based Liqueur

The distribution of OTUs among different samples was evaluated using a Venn diagram. The bacterial diversities in CGL-CRW-1, CGL-CRW-2, and CRW were relatively similar, with 57, 70, and 52 OTUs, respectively (Figure 6A,B). A total of 40 bacterial OTUs (47.06%) were shared among all samples. For fungi, the OTU distribution was as follows: 7 in CGL-CRW-1, 10 in CGL-CRW-2, and 15 in CRW, with 7 OTUs (43.75%) shared across all samples. Among the three, CGL-CRW-2 exhibited the highest total diversity and the most unique OTUs, approximately twice as many as those in CGL-CRW-1 and CRW. These findings indicate that the addition of Chinese gall leaven enhanced the overall microbial community diversity in Huangjiu.

Figure 6.

Venn plots of CGL-CRW and CRW according to the fungal (A) and bacterial (B) communities, with the evaluation of the genus level. Principal coordinate analysis of the fungal (C) and bacterial (D) genera in CGL-CRW and CRW. Two-dimensional chromatogram results (E) and gallery plot of volatile fingerprints (F) of aroma release differences in CGL-CRW and CRW.

To further explore the divergence in the microbial community structure among the three different samples, principal coordinate analysis was performed based on Binary-Jaccard and Bray–Curtis distances. The PCoA-based analysis of the fungal communities revealed that the first (PC1) and second (PC2) principal components respectively accounted for 43.2 and 25.1% of the variations between samples. For bacteria, the contribution rates of PC1 and PC2 were 73.2% and 26.8%, respectively. The distance among CGL-CRW-1, CGL-CRW-2, and CRW demonstrated a distinct separation trend, which showed significant differences in species diversity. Additionally, the different processing methods of Chinese gall leaven resulted in distinct microbial compositions in Chinese rice wine. Different microbial strains are critical to achieving the targeted diversification of Huangjiu products. For instance, Yarrowia lipolytica strains exhibiting high erythritol-producing capacity have been employed as adjunct cultures in Huangjiu fermentation, where they significantly enhance the cereal-derived aroma of the final product [43].

A heatmap analysis of bacterial and fungal diversity was conducted to compare microbial community composition across samples (Figure 2C,D). A total of 50 bacterial genera were present in high abundance. Among them, Pediococcus and Lactiplantibacillus were particularly abundant in CGL-CRW-1. Previous studies have shown that Weissella, Pediococcus, and Lactobacillus were closely associated with flavor compound production in rice wine [35]. Weissella and Lactobacillus, both lactate-producing bacteria, showed positive correlations with pH, amino acid nitrogen, and alcohol content. This aligns with the metabolic pattern of lactate-producing bacteria, which convert sugars into ethanol, degrade proteins into amino acids, and produce acidic compounds [44]. Notably, Lactobacillus can also produce acetic acid and various antimicrobial substances, including bacteriocins, which inhibit the growth of spoilage and pathogenic microorganisms [20]. The heatmap further showed significant variation in fungal composition among the samples. Rhizopus spp., known for producing various enzymes and metabolites that promote metabolic activity, is an important genus associated with flavor compound synthesis in Huangjiu [38]. Saccharomyces, with its strong ethanol-producing ability, plays a crucial role in fermentation. Both Rhizopus and Saccharomyces were found in relatively high abundance in CGL-CRW-1. Additionally, the non-Saccharomyces yeast Wickerhamomyces, which can synthesize enzymes to catalyze the formation of esters, acids, higher alcohols, and other flavor compounds, was abundant in CGL-CRW-2. The relative abundances of Saccharomyces and Wickerhamomyces were significantly elevated, which aligns well with the GC-MS results showing a marked increase in acetate esters (e.g., isoamyl acetate) and ethyl esters in the CGL-CRW samples. These esters are key contributors to the formation of pleasant fruity and floral aromas. During the Huangjiu fermentation, diverse microorganisms produce hundreds of metabolites that form its unique aroma, while the microbiome composition strongly influences the final product’s aroma and quality [3].

During the fermentation process of Chinese rice wine, the flavor metabolites and microbial community changed dynamically with the brewing process [47]. Therefore, Spearman’s correlation analysis was performed to assess the association between characteristic microorganisms and key flavor metabolites (Figure S3). Acetobacter_indonesiensis, Caulobacter_vibrioides, Collinsella_aerofaciens, Delftia_tsuruhatensis, Herbaspirillum_huttiense, Methylobacterium_brachiatum, Pantoea_dispersa, Pediococcus_pentosaceus, Rhodococcus_erythropolis, Romboutsia_ilealis, Saccharopolyspora_gregorii, Saccharopolyspora_rectivirgula, Saccharopolyspora_rectivirgula, Sphingobacterium_multivorum, Staphylococcus_caprae, Staphylococcus_sciuri, Thermoactinomyces_vulgaris, Aspergillus_penicillioides, Candida_tropicalis, and Cyberlindnera_fabianii showed significant positive correlations with ethyl phenylacetate, isoamyl hexanoate, capric acid isoamyl ester, palmitic acid ethyl ester, 3-methyl-1-butanol, 2,3-butanediol, 1-hexanol, 1-heptanol, 1-octen-3-ol, 4-Hydroxy-3-methoxystyrene, β-caryophyllene, and Alpha-caryophyllene, similar to the results of the aroma compounds. Based on the above findings, it can be inferred that certain microorganisms play a key role in the biosynthesis of various compounds. Furthermore, the production of specific aroma components may be attributed to synergistic interactions among multiple microbial species [48]. Notably, the relative abundances of Saccharomyces and Wickerhamomyces showed strong positive correlations with the concentrations of several acetate esters (e.g., isoamyl acetate) and phenethyl alcohol. These findings were corroborated by the sensory evaluation, where the pronounced fruity and floral characteristics observed in the CGL-CRW samples could be directly attributed to the enrichment of these yeast genera and the consequent activation of their ester-synthesis pathways. Furthermore, the relative abundances of Lactiplantibacillus and Pediococcus were positively correlated with the concentrations of lactic acid and certain organic acids, indicating their role in modulating the acidity of the fermentation system, which in turn contributed to the overall flavor balance and harmony. Principal component analysis was used to investigate the possible grouping of both sensory attributes and volatile compounds with an OAV > 1 that may contribute significantly to the overall aroma of the Chinese rice wine (Figure S4) [45]. Figure S4 showed that the first principal component (PC1) had the greatest influence and accounted for 68.9% of the variability, while the second principal component (PC2) accounted for 31.1% of the variability. And these three samples are well distinguished and distributed in different regions.

In summary, the addition of Chinese gall leaven enhanced the abundance of fungi such as Aspergillus, Saccharomyces, and Rhizopus, which are strongly associated with the flavor profile of Huangjiu. These microbial disparities thus provide a mechanistic link between CGL addition and the previously observed improvements in enzymatic activity and substrate utilization. The sample fermented with CGL-1 resulted in a profile with relatively stronger aldehyde signatures (Figure 5B) and sustained high activity of cell-wall degrading enzymes (β-glucosidase, xylanase) post-fermentation. This aligns with its microbial community, which was enriched in saccharifying and cellulolytic molds like Aspergillus and Rhizopus. These microbes are efficient at breaking down complex substrates, which may also liberate precursors for aldehyde formation via Strecker degradation or polyphenol oxidation. The early and high peak of acid protease activity in CRW-CGL-2 could generate more amino acid precursors, supporting both microbial growth and the synthesis of higher alcohols/esters via the Ehrlich pathway. These differentiated profiles suggest that CGL-1 might be preferred for developing Huangjiu with a more complex, potentially aged-like character (from aldehydes) and efficient fermentation, while CGL-2 could be chosen to maximize bioactive phenolic content and create a fruitier, more ester-driven aroma profile. This understanding provides a basis for tailoring functional Huangjiu products by selecting specific CGL sources as customized fermentation starters.

4. Conclusions

This study proposes a novel strategy for producing a novel low-alcohol, bioactive-enriched, and antioxidant-active functional Huangjiu-based liqueur by incorporating traditional fermentation agents such as Chinese gall leaven, which modulates the microbial and enzymatic profile to enhance both health-promoting properties and flavor complexity without compromising the base wine’s character. The study presents a potential approach for value-added product innovation that aligns with current market trends toward health-oriented, low-alcohol beverages. This approach aligns with the growing demand for healthier low-alcohol beverages and adds value to traditional fermented products. However, translation to industrial scale requires addressing challenges in microbial stability during scale-up, thorough safety, and regulatory evaluation. Future work should also focus on pilot-scale verification, toxicological assessment, in vivo functional testing, and storage stability to establish a solid foundation for commercialization.

Abbreviations

The following abbreviations are used in this manuscript:

CGL	Chinese gall leaven
CRW	Chinese rice wine
CRW-CGL-1	Chinese rice wine fermented with Chinese gall leave that obtained from Tongrentang Chinese Medicine Co., Ltd. (Beijing, China)
CRW-CGL-2	Chinese rice wine fermented with Chinese gall leave that obtained from Huqingyu Tang Pharmaceutical Co., Ltd. (Hangzhou, China)

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040739/s1.

Author Contributions

X.Z. (Xiaolei Zhu): Formal Analysis; Writing—Original Draft; M.J.: Methodology; Investigation; X.Z. (Xue Zhang): Investigation; Software; C.Z.: Data Curation; Writing—Review and Editing; Y.M.: Methodology; Investigation; J.Z.: Resources; Data Curation; B.Y.: Software; Writing—Review and Editing; Y.L.: Data Curation; Writing—Review and Editing; C.S.: Conceptualization; Funding Acquisition; T.X.: Project Administration; Supervision; X.X.: Conceptualization; Funding Acquisition; Project Administration; J.M.: Conceptualization; Resources. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

This study did not require ethical approval. In China, ethical review is not legally mandatory for sensory evaluations of low-risk foods. This study falls into this category. We recruited healthy volunteers to conduct routine tasting tests on low-risk foods. The entire process involved no invasive procedures, and the risk to participants was extremely low. No physical interventions, injections, or other procedures that could potentially harm participants were performed.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Author Yingying Mao was employed by the company China Shaoxing Yellow Rice Wine Group Co., Ltd. She participated in Methodology and Investigation in the study. Author Jiandi Zhou was employed by the company China Shaoxing Yellow Rice Wine Group Co., Ltd. He participated in the Resources and Data Curation in the study. The role of the company was to provide access to relevant industry resources, including specific raw materials and production facilities. The involvement of authors from the company had no any effect on the objectivity and authenticity of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Statement

This work was supported by the National Natural Science Foundation of China (32302040); Shaoxing Basic Science and Technology Special Project (2025A11017); Zhejiang Provincial Leading Goose Research and Development Plan (2024C02007); Natural Science Foundation of Zhejiang Province, China (LQ21C200003); Zhejiang University Student Science and Technology Innovation Activity Plan (Xinmiao Talents Plan) (S202510349071, 2024R431A010); National College Students Innovation and Entrepreneurship Training Program Project (202410349047).