Functional properties and flavor of fermented germinated brown rice-chickpea beverage with Lactobacillus plantarum

Abstract

This study developed a fermented beverage from germinated brown rice and chickpea using Lactobacillus plantarum. The results showed that the 36 h-fermented product exhibited strong antioxidant capacity (DPPH: 81.16 %; ABTS: 76.37 %; FRAP: 3.44 μmol/L) and significant inhibitory effects on α-amylase (69.94 %) and α-glucosidase (70.84 %), highlighting its potential antidiabetic properties. Although simulated in vitro digestion led to a reduction in biological activities of FGCB, its substantial antioxidant and antidiabetic activities were retained. Importantly, fermentation converted undesirable off-flavor-producing aldehydes into alcohols, ketones, and organic acids, among which 3-hydroxy-2-butanone, 1-hexanol, and 1-butanol were identified as key contributors to the distinctive flavor of FGCB. This study lays a foundation for the development and industrial production of new-type high-quality fermented beverages.

Highlights

• •A novel high-quality FGCB was produced by Lactobacillus plantarum.
• •FGCB had strong antioxidant and antidiabetic capacity.
• •Key flavor compounds in FGCB were 3-hydroxy-2-butanone, 1-hexanol, and 1-butanol.
• •After gastrointestinal digestion, FGCB still retained antioxidant and antidiabetic activities.

1. Introduction

In recent years, with the increasing prominence of health issues such as lactose intolerance and cow's milk allergy, coupled with ethical considerations regarding animal welfare, the global market demand for sustainable, hypoallergenic, and functional plant-based beverages has risen significantly; due to their core advantages of low cholesterol content, high dietary fiber content, and diverse bioactive substances, such beverages have become a key research direction in the field of food science (Demarinis et al., 2024). Moreover, probiotic fermentation not only improves the digestibility and gastrointestinal benefits of cereal beverages but also enhances their flavor and functionality by producing metabolites such as lactic acid, amino acids, and aromatic compounds, thus gaining increasing popularity among consumers. (Yang et al., 2022). As a “generally recognized as safe” and widely used lactic acid bacterium, Lactobacillus plantarum (L. plantarum) demonstrates outstanding probiotic efficacy and versatile metabolic activities, thus holding high value in the fields of food fermentation and health promotion. For instance, fermenting rice protein with L. plantarum NCU137 leads to a bacterial concentration of approximately 8.88 log CFU/mL under optimized conditions, while also improving flavor and antioxidant activity (Peng et al., 2024).

Germinated brown rice (GBR) is a product obtained by germinating brown rice to a definite sprout length under controlled temperature and humidity conditions; after germination, it not only exhibits significantly improved sensory properties and flavor, but also produces various bioactive components with health-promoting and disease-preventing effects, such as γ-aminobutyric acid (GABA) and oligosaccharides. Existing studies have confirmed that regular consumption of GBR helps prevent colon cancer and heart disease, and can also alleviate stress, inhibit cancer cell proliferation, and lower blood pressure (Ren et al., 2022). These core functional advantages are highly aligned with the current demand for healthy food in the market—nowadays, in addition to traditional rice products like rice cakes and rice noodles, the market for functional rice products (e.g., embryo-retained rice and selenium-enriched rice) has shown substantial growth, while products such as blood glucose-lowering porridge, germ rice paste, and germinated grain instant powder (rich in GABA) also hold a stable position in the market (Loan et al., 2025). This further confirms the market potential of functional rice products.

Chickpea (Cicer arietinum L.), is the third most highly consumed legume globally. Renowned for its high nutritional value, chickpea is rich in high-quality plant proteins, diverse essential amino acids, as well as flavonoids, steroids, and other bioactive components. They exhibit significant antioxidant, anti-inflammatory, and cholesterol-lowering properties and also hold broad application prospects in the development of functional foods (Wallace et al., 2016).

Based on the above context, this study aims to develop a functional beverage using GBR and chickpeas as raw materials via fermentation with L. plantarum, a research direction that has not been reported previously. Compared with previous fermented beverage systems using traditional grains or single legumes as raw materials, this study, on one hand, achieves complementary amino acid composition through the combination of grains and legumes, constructing a high-quality protein source with balanced nutrition; on the other hand, it relies on the synergistic adaptation of the two raw materials in terms of flavor and texture characteristics to significantly enhance the palatability and comprehensive functional properties of the fermented beverage. Furthermore, this study analyzes the antioxidant activity and antidiabetic potential of the beverage before and after in vitro digestion to comprehensively evaluate its actual health value, ultimately providing scientific support for the development of high-quality functional plant-based beverages.

2. Materials and methods

2.1. Materials and reagents

“Huruan 1212” brown rice was procured from the Zhuanghang Experimental Station of Shanghai Academy of Agricultural Sciences, chickpeas (6.43 % moisture, 21.2 % protein, 4.2 % fat, 60.1 % carbohydrates) were obtained from Huaye Liangtian Foods Co., Ltd. (Shandong, China), and L. plantarum BNCC194165 was purchased from Henan Beina Biological Inspection and Testing Co., Ltd. (Henan, China).

2.2. Cultivation of strain

L. plantarum BNCC1941165 was cultivated in de Man, Rogosa, and Sharpe (MRS) medium at 37 °C for 16 h. After centrifuging the culture for 5 min at 8000 rpm and rinsing it three times with a 0.9 % sterile saline solution, a culture with a cell density of approximately 7 log CFU/mL was obtained. This served as the inoculum for grain fermentation.

2.3. Germination of brown rice

The brown rice was first rinsed three times and then sterilized by soaking in a 1 % sodium hypochlorite solution for 10 min. Subsequently, it was washed five times with sterile water and soaked at 25 °C for 12 h. The brown rice was then transferred to a constant-temperature incubator (35 °C, 70 % relative humidity) for 24 h to promote germination. Afterwards, enzyme inactivation was achieved by hot-water treatment (100 °C) for 10 min. Finally, the sample was desiccated at 55 °C for 10 h. The nutritional profile of GBR was as follows: 9.17 % moisture, 6.68 % protein, 2.1 % fat, and 85.89 % carbohydrates.

2.4. Preparation of the fermented germinated brown rice-chickpea beverage (FGCB)

Preliminary experiments indicated that the incorporation of chickpeas enhanced the protein content of the fermented drink. Based on sensory evaluations, the optimal amount of chickpea addition was determined to be 10 %.

The GBR-chickpea mixture was immersed in distilled water at a ratio of 1:5 for 12 h and then homogenized into a slurry. The rice slurry was gelatinized at 90 °C for 30 min. Then, 0.1 % α-amylase (4000 U/g) and 0.1 % glucoamylase (100,000 U/g) were added, and the enzymatic hydrolysis was conducted at 65 °C for 30 min. The beverage was then sterilized at 121 °C for 10 min to inactivate the enzymes. After cooling to ambient temperature (25 °C), 5 % (v/v) L. plantarum was added as an inoculum, and fermentation was carried out at 37 °C for 48 h (as shown in Fig. 1). Samples were collected for microbiological and chemical analysis at 0 h, 12 h, 24 h, 36 h, and 48 h.

Fig. 1.

Process Flow for FGCB.

2.5. Physicochemical indicators

2.5.1. Determination of pH, titratable acidity (TA), total soluble solids (TSS), and viable cell count

The pH values of the fermented beverages during various fermentation phases were measured using a pH meter (Leici DZS-706, China). Meanwhile, TA was determined via titration against 0.1 M NaOH, using phenolphthalein as the indicator. The findings were presented as the percentage of lactic acid (Wang et al., 2019). In addition, the TSS content of the samples was analyzed using an automatic Abbe refractometer (Shanghai Ino, China), and the results were expressed as °Brix values. Finally, viable cell counts were determined by enumerating the colony-forming units (CFU) on MRS agar. The fermented beverage was subjected to serial of ten-fold gradient dilution using 0.9 % sterile saline, and the diluted samples (0.1 mL) were inoculated onto MRS agar plates and incubated at 37 °C for 48 h. Colony counts were recorded and expressed as log CFU/mL.

2.5.2. Nutrient content analysis

The protein and fat content of the fermented beverages was assessed using the bicinchoninic acid (BCA) and acid hydrolysis methods (GB 5009.6–2016), respectively. The reducing sugar content was measured using the DNS colorimetric method, following the procedure described by Guo et al. (2024).

Meanwhile, the GABA content was analyzed by high-performance liquid chromatography (HPLC) as described by Wu et al. (2022) with slight modifications. Briefly, 0.5 mL of each sample was combined with 0.1 mL of a NaHCO₃ solution and 0.2 mL of 4-dimethylaminoazobenzene-4‑sulfuryl chloride. The procedure was carried out at 70 °C for 20 min, followed by filtration. The HPLC system was equipped with a Compass C18 reversed-phase chromatographic column (250 mm × 4.6 mm, 5 μm).

The total phenol content (TPC) was evaluated using the Folin-Ciocalteu method based on the procedure reported by Pham et al. (2024). Briefly, 0.1 mL of diluted solution was mixed with 0.5 mL of the Folin-Ciocalteu reagent and 0.4 mL of a 7.5 % Na₂CO₃ solution. This mixture was incubated in the dark at 40 °C for 40 min, and its absorbance was subsequently measured at 765 nm with an Infinite 200 PRO multimode microplate reader (Tecan, Switzerland). The TPC was expressed as milligrams of gallic acid equivalents (GAE) per 100 mL.

The colorimetric method outlined by Joseph Bassey et al. (2024) was employed to assess the total flavonoid content (TFC). Briefly, 1 mL of the diluted sample was mixed with 0.3 mL of a 5 % NaNO₂ solution and incubated at room temperature for 6 min. Then, 0.3 mL of a 10 % AlCl₃ solution was added, followed by thorough mixing and another 6-min incubation. Following the addition of 1 M NaOH (2 mL), the mixture was incubated in the dark for 10 min. The absorbance was finally measured at 510 nm. The TFC was expressed as milligrams of rutin equivalents (RE) per 100 mL.

2.6. Antioxidant activity

2.6.1. DPPH and ABTS radical scavenging

The DPPH radical scavenging rate of the beverage samples was evaluated as reported by Jiang et al. (2025). Briefly, 1 mL of each sample was mixed with 1 mL of a DPPH-ethanol solution (0.1 mmol/L) and then incubated for 30 min away from light. Finally, the absorbance at 517 nm was evaluated.

The ABTS stock solution was prepared as described by Tyagi et al. (2022). Briefly, 100 μL of the sample was mixed with 1 mL of the ABTS radical solution and incubated at ambient temperature for 40 min under dark conditions. Finally, the absorbance was measured at 734 nm. The following formula was used to calculate the DPPH/ABTS radical scavenging activity.

DPPH/ABTS radical scavenging activity (%) = (1–(A_s–A_b)/A_c) × 100 %.

Here, A_s: absorbance of the sample mixed with DPPH/ABTS; A_b: absorbance of the sample mixed with distilled water; and A_c: absorbance of distilled water mixed with DPPH/ABTS.

2.6.2. Ferric antioxidant power (FRAP)

The FRAP reagent was prepared according to the method reported by Ziarno et al. (2024). The FRAP working reagent was freshly prepared by combining 10 mM tripyridyltriazine in 40 mM HCl, 20 mM FeCl₃, and 0.3 M acetate buffer (1:1:10, v/v/v). This mixture was placed at 37 °C. Subsequently, 50 μL of each sample was mixed with 1.5 mL of the FRAP solution and incubated at 37 °C for 10 min. Finally, the absorbance was measured at 593 nm.

2.7. Antidiabetic potential

2.7.1. α-Amylase inhibitory activity

The α-amylase activity inhibitory activity was determined following the method reported by Jiang et al. (2024). Briefly, 1 mL of each sample solution was mixed with 1 mL of α-amylase (0.6 mg/mL) and 1 mL of starch (2.0 mg/mL) before incubation at 37 °C for 20 min. Afterwards, 2 mL of DNS was added, and the absorbance was evaluated at 540 nm following 5 min of boiling in a water bath.

2.7.2. α-Glucosidase inhibitory activity

The α-glucosidase inhibition rate was detected based on the protocol reported by Jiang et al. (2024), with minor modifications. Briefly, 100 μL of each sample solution was mixed with 100 μL of α-glucosidase (0.01 mg/mL) and reacted at 37 °C for 15 min. This was followed by the addition of 200 μL 4-nitrophenyl α-D-glucopyranoside (ρNPG, 2.5 mmol/L), and the reaction was carried out in a water bath at 37 °C for 10 min. Subsequently, the reaction was terminated via the addition of 1 mL Na₂CO₃ (0.1 mol/L), and the absorbance was evaluated at 410 nm.

2.8. In vitro digestive simulation

Following a standardized in vitro digestion protocol (Brodkorb et al., 2019), samples were subjected to sequential gastric and intestinal digestion. The collected digesta were inactivated in a boiling water bath for 5 min and then centrifuged at 8000 rpm for 5 min. Subsequently, the supernatant was employed to analyze the antioxidant activity and antidiabetic potential of the samples.

2.9. Gas chromatography–ion mobility spectrometry (GC-IMS) analysis

The volatile compounds in FGCB were analyzed using GC-IMS (FlavourSpecR, Dortmund, Germany) following the procedure reported by Song et al. (2025), with minor modifications. Exactly 1 mL of each sample was placed in a 20 mL headspace vial, and 10 μL of 2-octanol (100 mg/L) was added. The mixture was then incubated at 60 °C for 15 min at 500 rpm. Subsequently, 0.5 mL of the sample mixture was injected onto a capillary column (15 m × 0.53 mm, 1.0 μm, Restek, USA) for chromatographic separation. The column temperature was set at 60 °C, and high-purity nitrogen (purity >99.999 %) was used as the carrier gas. The temperature of the detector (IMS) was set to 45 °C. The analytical procedure followed the following flow rate program: initial flow rate of 2.0 mL/min for 2 min, linear increase to 10.0 mL/min over 8 min, linear increase to 100 mL/min over 10 min, and then held at 100 mL/min for 10 min. The ion mobility spectra were obtained in the positive ion mode using a 3H ion source at a flow rate of 150 mL/min.

2.10. Sensory evaluation of FGCB

Sensory evaluation was conducted in accordance with the methodology detailed by Yu et al. (2024). Sensory analyses were carried out by 20 professionally trained evaluators. All samples were placed in transparent glass cups labeled with random three-digit numbers and provided to evaluators in a random and monadic way. To minimize carry-over effects, evaluators were asked to cleanse their palates with water before evaluating each sample. The tested sensory attributes included color, odor, smoothness, sourness, sweetness, rice aroma, beany odor, and overall acceptability. A scale of 1–10 (1 = poor, 10 = excellent) was used to assess the subjective preference of the evaluators for the product's characteristics. All samples were served at room temperature and randomly evaluated in triplicate, and the average was recorded as the result.

2.11. Statistical analysis

All experiments were conducted in triplicate unless otherwise specified. Data analysis was performed using IBM SPSS Statistics 26 (SPSS Inc., Chicago, USA). The statistical significance of differences among groups (0, 12, 24, 36, and 48 h) was evaluated using one-way ANOVA, followed by Duncan's test. The significance level was set at p < 0.05. Origin 2021 software was used for plotting graphs.

3. Results and discussion

3.1. Physicochemical properties of FGCB

pH and TA not only serve as key indicators of fermentation progress but are also crucial determinants of beverage taste and quality. L. plantarum produces acids such as lactic acid and acetic acid during the fermentation process, leading to a decrease in pH and an increase in acidity. In this study, the pH of FGCB decreased rapidly from 6.32 ± 0.03 to 3.83 ± 0.02 at 12 h of fermentation and then showed a slow decrease to 3.33 ± 0.02 at 48 h (Table 1). This pattern was likely observed because the optimal pH range for the growth of L. plantarum is 4.5–6.8. During the initial stage of fermentation, L. plantarum proliferated rapidly, leading to a steep increase in acidic compounds. However, as fermentation progressed, the rising acidity of the system gradually inhibited the growth and reproduction of L. plantarum, resulting in pH stabilization. In addition, during the 48-h fermentation period, the TA of FGCB increased significantly from 0.83 ± 0.11 g/L to 8.93 ± 0.11 g/L (p < 0.05; Table 1), and this trend was consistent with the decreasing trend of pH. These findings could be attributed to the metabolic activities of L. plantarum in the fermented beverage.

Table 1.

Changes in the physicochemical indicators and nutrient content of FGCB at different fermentation time points.

Parameters	0 h	12 h	24 h	36 h	48 h
pH	6.32 ± 0.03e	3.83 ± 0.02d	3.55 ± 0.01c	3.40 ± 0.02b	3.33 ± 0.02a
Titratable acidity (g/L lactie acid)	0.83 ± 0.11a	4.32 ± 0.04b	6.75 ± 0.21c	7.95 ± 0.21d	8.93 ± 0.11e
Viable cell count (Log CFU/mL)	6.03 ± 0.11a	7.39 ± 0.03b	7.40 ± 0.05b	7.40 ± 0.00b	7.42 ± 0.03b
TSS (°Brix)	11.75 ± 0.06b	11.57 ± 0.01b	11.34 ± 0.02a	11.31 ± 0.03a	11.28 ± 0.04a
Protein (mg/mL)	4.56 ± 0.13a	4.56 ± 0.06a	4.62 ± 0.11a	5.85 ± 0.23b	6.09 ± 0.14b
Fat (%)	1.25 ± 0.07d	0.98 ± 0.03c	0.77 ± 0.04b	0.62 ± 0.03a	0.59 ± 0.01a
Reducing sugars (mg/mL)	81.05 ± 1.67b	96.03 ± 8.39c	64.98 ± 0.72a	62.42 ± 7.89a	58.33 ± 4.22a
GABA (mg/100 mL)	6.32 ± 0.10a	6.99 ± 0.14b	7.09 ± 0.02b	8.15 ± 0.13c	8.17 ± 0.16c
TPC (mg GAE/100 mL)	123.42 ± 2.34a	136.48 ± 2.23b	146.57 ± 1.03c	164.37 ± 1.71e	155.98 ± 1.16d
TFC (mg RE/100 mL)	283.75 ± 18.56a	433.13 ± 24.75b	550.00 ± 16.79c	613.75 ± 2.65d	573.75 ± 15.03cd

The viable cell count is a key indicator of fermentation quality. Although there is no definitive consensus on the minimum number of probiotic bacteria required to achieve health benefits in the host, it is generally accepted that the final product should contain at least 6–7 log CFU/mL of viable probiotic cells (Montanari et al., 2020). As shown in Table 1, within the first 12 h of fermentation, L. plantarum grew rapidly, with its viable count increasing from 6.03 ± 0.11 log CFU/mL to 7.39 ± 0.03 log CFU/mL. During the latter phase of fermentation, however, the growth rate of L. plantarum decreased, but the viable count reached 7.40 ± 0.00 log CFU/mL at 36 h.

The dynamic changes in the TSS content reflect the metabolic activity of lactic acid bacteria and the efficiency of substrate conversion. Table 1 shows that the TSS content of FGCB showed a slight decrease from 11.75 ± 0.06 to 11.34 ± 0.02 within 24 h of fermentation. This was due to the consumption of sugars by L. plantarum and their conversion to lactic acid during the early stage of fermentation. Notably, the TSS content of FGCB stabilized during the later stages and eventually declined to 11.28 ± 0.04 (48 h).

3.2. Changes in the nutrient content of FGCB

As shown in Table 1, the protein content of FGCB remained largely unchanged during the initial phase of fermentation. However, it increased in the later phase, reaching 6.09 ± 0.14 mg/mL. This increase was attributed to the release of soluble proteins via the action of microbial enzymes and the synthesis of bacterial proteins by L. plantarum during proliferation. These observations were consistent with reports from Hurtado-Murillo et al. (2025), who found that the probiotic fermentation of quinoa-chickpea enhanced the total protein content (2.2–2.3 %). Notably, the fat content of FGCB decreased from 1.25 % (0 h) to 0.59 % (48 h) (Table 1), likely due to the lipid-degrading activity of L. plantarum (Yu et al., 2023).

In addition, the reducing sugar content of FGCB increased from 81.05 ± 1.67 mg/mL to 96.03 ± 8.39 mg/mL during the first 12 h of fermentation but showed a significant decrease thereafter (p < 0.05), reaching 58.33 ± 4.22 mg/mL by 48 h (Table 1). This change was due to the early metabolism of saccharification products such as maltose, which initially led to an increase in the levels of reducing sugars. However, during the subsequent growth of L. plantarum, the sugars were consumed and broken down into organic acids, thereby affecting the reducing sugar levels (Tu et al., 2024).

GABA, as an inhibitory neurotransmitter, possesses numerous well-recognized physiological functions. For instance, GABA can regulate blood pressure, improve cell metabolism, and reduce cholesterol levels. Moreover, GABA has shown potential therapeutic applications in the treatment of neurological diseases and cancer (Tufail et al., 2025). As shown in Table 1, GABA content of FGCB progressively increased from 6.32 ± 0.10 mg/100 mL (0 h) to 8.17 ± 0.16 mg/100 mL (48 h) as fermentation advanced. This increase was attributed to the ability of L. plantarum to convert L-glutamic acid to GABA via glutamic acid decarboxylase (GAD). The decrease in the rate of GABA synthesis during the later stages of fermentation was likely due to the accumulation of primary fermentation products such as lactic acid, which lowered the pH value and thus affected the conformation of GAD, electrolyzing the side chain groups and reducing its activity (Lu et al., 2025).

3.3. Changes in the TPC and TFC of FGCB

Fermentation by lactic acid bacteria facilitates the conversion and release of phenolic compounds from grains and enhances their bioavailability (Islam et al., 2024). As illustrated in Table 1, the TPC and TFC of the fermented beverages initially increased and subsequently declined, peaking at 164.37 ± 1.71 mg GAE/100 mL and 613.75 ± 2.65 mg RE/100 mL, respectively, at the 36 h point. These levels were 33.18 % and 116.30 % higher, respectively, than those in the unfermented sample. This increase was attributed to the production of hydrolytic enzymes such as cellulase, esterase, and β-glucosidase by L. plantarum during the fermentation process. These enzymes promoted the decomposition of macromolecular compounds into small-molecule polyphenol monomers, thereby increasing the overall level of phenolic substances (Zhu et al., 2020). Additionally, the release of bound phenolics from the plant cell wall could also increase the TPC and TFC (Li et al., 2021). Notably, at 48 h of fermentation, the TPC and TFC of FGCB decreased to 155.98 ± 1.16 mg GAE/100 mL and 573.75 ± 15.03 mg RE/100 mL, respectively, likely due to the acidic environment, which reduced the activity of hydrolytic enzymes that produce small molecules, such as phenolic acids (Tu et al., 2024). Another possible contributing factor was the covalent bonding of phenols with the proteins, peptides, and organic acids produced during fermentation (Chen et al., 2025). Similar phenomena were reported in the study of mixed grain fermentation by Ren et al. (2025).

3.4. Changes in the antioxidant activity of FGCB

Phenolic and flavonoid compounds possess strong antioxidant properties, and they can efficiently scavenge free radicals and reactive oxygen species. Thus, they can help in preventing cancer, diabetes, and neurodegenerative diseases. In this study, the DPPH (Fig. 2A) and ABTS (Fig. 2B) radical scavenging abilities of FGCB and its FRAP (Fig. 2C) increased significantly (p < 0.05) during the first 36 h of fermentation. During this period, the DPPH scavenging activity increased from 40.98 ± 1.97 % to 81.16 ± 2.54 % (1.98-fold), but decreased to 67.27 ± 2.29 % after 48 h of fermentation. Meanwhile, the ABTS radical scavenging rate reached 76.37 ± 2.12 % at 36 h and then slightly decreased to 71.05 ± 2.82 % at 48 h. In contrast, the FRAP increased gradually during fermentation, reaching 3.48 ± 0.16 μmol/mL at 48 h, this value was 34.36 % higher than that in the unfermented sample. Previously, similar results were reported by Fan et al. (2025). In their study, L. plantarum fermentation of chickpea milk significantly increased its ABTS and DPPH radical scavenging rates to 82.10 % and 73.72 %, respectively.

Fig. 2.

Effects of digestion on FGCB at different fermentation time points. (A) DPPH radical scavenging activity; (B) ABTS radical scavenging activity; (C) FRAP; (D) Correlation analysis for TPC, TFC, and antioxidant activities; (E) α-Amylase inhibitory activity; (F) α-Glucosidase inhibitory activity.

Note: Different lowercase letters indicate significant differences among fermentation time points, while different uppercase letters denote significant differences among digestion stages (p < 0.05).

To explore the relationships between the antioxidant indicators, Pearson's correlation analysis was performed as shown in Fig. 2D, a significant (p < 0.05) positive correlation was detected between TPC and ABTS, DPPH, and FRAP (r = 1.00, 0.91, and 0.96, respectively), as well as between TFC and these antioxidant indexes (r = 0.95, 0.95, and 0.98, respectively). Hence, the enhanced antioxidant activity of FGCB was primarily attributed to the enzymatic degradation of dietary fiber by microbes during fermentation, which converted bound phenolic compounds into free compounds with higher bioavailability. Additionally, the substances produced by L. plantarum, such as bacteriocins and exopolysaccharides, could also potentially scavenge free radicals, further enhancing the antioxidant capacity of FGCB (Mao et al., 2023).

3.5. Antidiabetic potential of FGCB

Type 2 diabetes mellitus (T2D) is a prevalent chronic metabolic disorder that not only poses a direct threat to patient health but also significantly increases the risk of mortality by triggering severe complications such as diabetic nephropathy and atherosclerotic cardiovascular diseases (Rangel-Galván et al., 2024). Inhibiting α-amylase and α-glucosidase can reduce the digestion of starch and oligosaccharides, thereby lowering postprandial blood glucose levels. Thus, this strategy is commonly used for preventing and controlling diabetes.

Previous studies have shown that natural plant-based products can effectively inhibit α-amylase and α-glucosidase following fermentation and thus lower blood sugar levels (Lai et al., 2024). In the present study, the inhibition rates of FGCB against α-amylase and α-glucosidase peaked at 74.91 % (24 h) and 70.84 % (36 h), respectively (Fig. 2E, Fig. 2F). In previous studies, the inhibitory rates of a probiotic GABA beverage against α-amylase and α-glucosidase were reported to be 61.97 % and 64.50 % respectively (Kittibunchakul et al., 2021). Meanwhile, the Lactobacillus helveticus-fermented milk with ultrasound-assisted exerted inhibition rates of 47.2 % against α-glucosidase and 43.4 % against α-amylase (Gholamhosseinpour et al., 2025). Thus, compared to other previously reported functional products, FGCB demonstrated higher α-amylase and α-glucosidase inhibitory activities. Notably, the antidiabetic effect of FGCB could partially be attributed to its phenolic compounds, such as p-coumaric acid and ferulic acid, which are natural α-glucosidase inhibitors. They achieve inhibition primarily through hydrogen bonding, hydrophobic forces, and ionic interactions with α-glucosidase (Ye et al., 2022). Furthermore, the proteolytic enzymes secreted by lactic acid bacteria can degrade proteins into bioactive peptides, which can inhibit α-amylase and α-glucosidase through structural interactions such as competitively binding to the active sites or allosteric modulating of the enzyme conformation (Kittibunchakul et al., 2021).

3.6. Changes in the antioxidant and antidiabetic activities of FGCB during in vitro digestion

During digestion, changes in pH, enzyme activity, and gut microbiota metabolism can alter the contents and bioavailability of bioactive compounds in different parts of the gastrointestinal tract. Assessing the changes in the antioxidant and antidiabetic activities of a beverage following simulated gastrointestinal digestion can thus demonstrate its nutritional value and stability. In this study, FGCB at different fermentation points showed a decrease in antioxidant properties following gastrointestinal digestion. Notably, the DPPH radical scavenging rate (Fig. 2A) of 36-h FGCB decreased from 81.16 ± 2.54 % to 58.95 ± 3.14 %, the ABTS radical scavenging rate (Fig. 2B) dropped from 76.37 ± 2.12 % to 63.25 ± 0.17 %, and the FRAP (Fig. 2C) reduced from 3.44 ± 0.31 μmol/L to 0.84 ± 0.05 μmol/L. This attenuation could be due to the binding of polyphenols to pepsin through hydrogen bonding, hydrophobic interactions, and van der Waals forces during gastric digestion (Zhao et al., 2023). In addition, during intestinal digestion, the increase in pH may have led to the destabilization of phenolic substances, and the digestive enzymes may have caused phenolic degradation, thus collectively reducing the antioxidant activity (Ziółkiewicz et al., 2023). Nevertheless, the digested products still retained significant DPPH and ABTS radical scavenging activity (∼50 %). This was likely due to the synergistic antioxidant effects of stabilized short-chain phenolic acids (p-coumaric acid, ferulic acid, etc.) generated during fermentation, along with the new peptides produced via enzymatic proteolysis (Sarmadi & Ismail, 2010). Notably, both the DPPH and ABTS radical scavenging capacities were the highest in 36-h fermented beverages before and after digestion.

The inhibitory effects of FGCB on α-amylase and α-glucosidase were reduced significantly after gastrointestinal digestion, as shown in Fig. 2E and Fig. 2F (p < 0.05). In FGCB subjected to 36 h of fermentation, the α-amylase inhibition rate decreased from 69.94 ± 2.41 % to 52.35 ± 1.45 %, while the α-glucosidase inhibition rate declined from 70.84 ± 11.86 % to 43.02 ± 2.87 % following digestion. This reduction was likely because the hydroxyl groups of phenolic compounds interacted with the amino acid residues in the enzyme active site through hydrogen and hydrophobic bonding, thereby inhibiting enzyme activity (Jiang et al., 2024). Additionally, the digestive environment and the presence of microbial enzymes may have led to the destabilization of phenolic compounds, diminishing their inhibitory effect on the target enzymes. These findings are consistent with the study by Jiang et al. (2025), who reported that the α-amylase and α-glucosidase inhibitory effects of polyphenols derived from peanut shells are weakened after in vitro digestion.

3.7. Changes in the volatile compounds of FGCB

The popularity of plant-based beverages often depends on their flavor, which is—in turn—affected by fermentation. Therefore, the volatile components of FGCB fermented for different durations were analyzed using GC-IMS. The qualitative spectral profiles of these compounds are illustrated in Fig. 3A. The principal component analysis (PCA) (Fig. 3B) revealed the clear separation of volatile metabolites across different fermentation phases (PC1: 63 %, PC2: 21 %), and tight intragroup clustering was also observed. This indicated that L. plantarum fermentation substantially impacts flavor development in cereal-based beverages.

Fig. 3.

Analysis of volatile compounds in FGCB at different fermentation time points. (A) Comparison of chromatograms; (B) PCA score plot; (C) Relative contents of various classes of volatile flavor compounds; (D) Fingerprint spectra.

A total of 34 volatile flavor components were identified during FGCB fermentation, including 11 aldehydes, 7 ketones, 7 alcohols, 4 esters, 2 acids, 1 furan, 1 terpene, and 1 ether. Compared to unfermented samples, the fermented samples showed a decrease in the relative content of aldehydes and a corresponding increase in alcohols, ketones, and acids (Fig. 3C). This suggested that L. plantarum converted aldehydes into alcohols, ketones, and acids, thus contributing to the overall flavor of FGCB.

Significant differences in volatile components were also observed across the different fermentation time points, as shown in Fig. 3D. Notably, the unfermented samples exhibited high levels of hexanal, heptanal, nonanal, pentanal, 1-penten-3-ol, and ethyl hexanoate (Table 2). Aldehydes such as hexanal, heptanal, nonanal, and pentanal, the main flavor substances in chickpea, can impart undesirable astringent and beany flavors (Tangyu et al., 2021). Notably, the aldehyde content was significantly reduced after fermentation (p < 0.05), especially at the 24 h and 36 h time points (Fig. 3C), which corresponded to the lowest concentration of aldehydes. This indicated that fermentation can effectively mitigate the negative impact of aldehydes on flavor.

Table 2.

Changes in the concentration of volatile flavor substances in FGCB at different fermentation time points.

NO.	Volatile flavor compounds	Concentration (μg/L)					Aroma description
		0 h	12 h	24 h	36 h	48 h
Aldehydes
1	Nonanal	44.73 ± 0.69d	13.90 ± 0.11c	11.05 ± 0.41a	11.48 ± 0.69ab	12.37 ± 0.64b	rose, citrus, strong oily
2	Benzaldehyde	6.67 ± 0.06a	6.27 ± 0.12a	8.00 ± 0.48b	7.68 ± 0.25b	7.55 ± 0.30b	bitter almond, cherry, nutty
3	Heptanal	43.57 ± 0.85c	16.51 ± 0.28a	15.96 ± 0.21a	15.72 ± 0.06a	18.4 ± 0.57b	fresh, fatty, green herbs, wine, fruity
4	Hexanal	203.67 ± 5.08c	39.31 ± 1.46b	32.87 ± 2.05a	33.06 ± 1.00a	36.53 ± 3.41ab	fresh, green, fat, fruity
5	Pentanal	5.16 ± 0.46b	1.14 ± 0.14a	0.86 ± 0.05a	0.93 ± 0.06a	1.20 ± 0.14a	green grassy, faint banana, pungent
6	(E)-2-Heptenal	27.17 ± 0.85a	34.53 ± 1.84b	33.80 ± 0.89b	37.46 ± 1.05c	39.25 ± 0.35c	spicy, green vegetables, fresh, fatty
7	(E, E)-2,4-Hexadienal	1.50 ± 0.24a	1.43 ± 0.13a	2.29 ± 0.16b	1.93 ± 0.19b	3.28 ± 0.25c	sweet, green, floral, citrus
8	(Z)-2-Hexenal	1.77 ± 0.41b	1.18 ± 0.10a	1.19 ± 0.11a	1.18 ± 0.16a	1.29 ± 0.05a	null
9	3-Methyl-2-butenal	5.80 ± 0.16a	5.86 ± 0.65a	6.92 ± 0.51b	7.57 ± 0.37b	7.22 ± 0.28b	fruity
10	2-Methylpentanal	5.80 ± 0.18d	5.46 ± 0.11c	2.82 ± 0.15a	3.37 ± 0.11b	6.66 ± 0.26e	vegetables, green, grapes
11	(E)-2-Butenal	11.64 ± 0.03b	12.22 ± 0.49b	11.61 ± 0.40b	10.71 ± 0.38a	10.38 ± 0.19a	null
Ketones
12	2-Heptanone	22.29 ± 0.89a	22.46 ± 0.61ab	22.70 ± 0.71ab	24.04 ± 0.99b	28.34 ± 1.04c	pear, banana, fruity, slight medicinal fragrance
13	3-Hydroxy-2-butanone	63.28 ± 2.64a	238.62 ± 14.31b	303.28 ± 8.15c	402.30 ± 19.30d	425.01 ± 11.87d	butter, cream
14	2-Butanone	17.07 ± 1.32a	30.14 ± 1.07b	40.09 ± 1.45c	39.44 ± 4.28c	38.29 ± 1.08c	fruity, camphor
15	2-Propanone	84.70 ± 1.19a	87.34 ± 1.72a	116.59 ± 1.65b	131.07 ± 1.06d	123.64 ± 2.28c	fresh, apple, pear
16	2-Octanone	4.64 ± 0.20b	4.17 ± 0.35a	4.17 ± 0.11a	4.26 ± 0.30ab	3.99 ± 0.06a	ketone, milk, cheese, mushroom
17	3-Hexanone	18.97 ± 1.65a	19.70 ± 2.95a	18.25 ± 1.18a	19.80 ± 2.76a	22.22 ± 3.27a	fruity, grape, sweet, rum
18	2-Pentanone	8.74 ± 0.33a	10.64 ± 0.26b	8.82 ± 0.16a	8.99 ± 0.16a	10.18 ± 0.55b	acetone, fresh, sweet fruity, wine
Alcohols
19	1-Hexanol	77.49 ± 5.85a	144.40 ± 2.66b	152.27 ± 3.39bc	156.87 ± 4.10c	180.11 ± 5.29d	fresh, fruity, wine, sweet, green
20	1-Butanol	243.09 ± 0.71a	277.13 ± 2.71b	302.37 ± 5.46c	310.18 ± 1.08d	301.04 ± 2.48c	wine
21	(Z)-2-Pentenol	7.52 ± 0.41a	8.27 ± 0.97a	11.19 ± 0.09b	13.10 ± 0.57c	12.77 ± 0.51c	green, plastic, rubber
22	Heptanol	4.42 ± 0.28a	7.13 ± 0.33b	7.57 ± 0.34b	7.60 ± 0.21b	8.23 ± 0.30c	herb
23	1-Octen-3-ol	6.85 ± 0.12a	10.95 ± 0.70b	11.68 ± 0.37c	11.81 ± 0.26c	12.61 ± 0.24d	mushroom, lavender, rose, hay
24	1-Pentanol	16.53 ± 0.36a	19.35 ± 0.36bc	19.37 ± 0.32bc	18.75 ± 0.20b	19.58 ± 0.54c	balsamic
25	1-Penten-3-ol	4.29 ± 0.03d	3.79 ± 0.07b	4.18 ± 0.16cd	3.99 ± 0.19bc	3.53 ± 0.10a	ethereal, green, tropical fruity
Acids
26	2-methylpropanoic acid	58.04 ± 8.35a	229.18 ± 45.63b	313.81 ± 12.76c	327.90 ± 12.10c	319.40 ± 5.69c	yogurt, rancid cream
27	Propanoic acid	0.80 ± 0.16a	4.15 ± 0.16b	4.34 ± 0.66bc	4.71 ± 1.04bc	5.28 ± 0.14c	yogurt, vinegar
Esters
28	Ethyl hexanoate	10.74 ± 0.49b	9.04 ± 0.28a	9.67 ± 0.45a	9.54 ± 0.62a	8.99 ± 0.34a	pineapple, fruity, wine
29	ethyl 2-(methylthio)acetate	4.55 ± 0.36a	9.64 ± 1.13b	11.20 ± 0.31c	12.80 ± 0.64d	12.57 ± 0.13d	Fruit, Green, Onion, Sulfur
30	Ethyl 2-methylpentanoate	4.05 ± 0.15a	4.72 ± 0.30b	4.08 ± 0.21a	5.26 ± 0.31c	5.65 ± 0.19c	fresh fruit flavor, cucumber, apple peel, pineapple
31	Acetic acid butyl ester	2.81 ± 0.25c	2.39 ± 0.10b	2.01 ± 0.04a	2.25 ± 0.02ab	2.29 ± 0.17b	fruity
Furans
32	2-Pentyl furan	35.33 ± 0.66ab	35.12 ± 1.02a	36.68 ± 0.53bc	36.18 ± 0.86abc	37.46 ± 0.81c	bean, fruity, earthy, green, vegetable
Terpenes
33	β-Pinene	5.09 ± 0.23d	4.28 ± 0.18c	3.02 ± 0.06b	2.45 ± 0.13a	2.42 ± 0.25a	resin, green
Ethers
34	tert-Butyl methyl ether	35.33 ± 0.50b	36.81 ± 0.78b	10.01 ± 1.14a	11.77 ± 0.29a	45.16 ± 1.69c	fruity, ether

In addition, the concentrations of ketones (3-hydroxy-2-butanone, 2-butanone, 2-propanone, 2-heptanone, and 2-pentanone, etc.), alcohols (1-hexanol, 1-butanol, heptanol, 1-octen-3-ol, and 1-pentanol, etc.), and acids (2-methylpropionic acid) significantly increased in the final phase of fermentation (p < 0.05). Ketones and alcohols could act as key contributors to the creamy, sweet, and fruity aromas of FGCB. Notably, among the ketones, 3-hydroxy-2-butanone (Acetoin) (odor threshold: 14 μg/L) showed a marked increase in content from 63.28 ± 2.64 μg/L to 425.01 ± 11.87 μg/L after 48 h of fermentation (p < 0.05), adding a distinct creamy and buttery aroma to the final product (Ren et al., 2025). Meanwhile, the levels of 2-propanone peaked at 131.07 ± 1.06 μg/L after 36 h of fermentation, and this compound provided a crisp and faint fruity aroma. In addition, the concentration of 1-hexanol (odor threshold: 5.6 μg/L), a common metabolite of L. plantarum, increased significantly after fermentation (p < 0.05; Table 2). Interestingly, previous studies have shown that 1-hexanol is an important flavor compound in fermented dairy products, imparting a delicate aroma and soft flavor (Peng et al., 2024).

The starch in grains is decomposed into fermentable sugars by enzymes secreted by microorganisms and then metabolized into the corresponding acids. In this study, propanoic acid, an important short-chain fatty acid, showed a peak concentration of 5.28 ± 0.14 μg/L after fermentation, enhancing the faintly sour aroma of FGCB (Table 2). Meanwhile, tert-butyl methyl ether, an unwanted flavor-generating compound that not only reduces sensory attributes but also presents potential health hazards, showed a significant reduction (p < 0.05) during the 24-h and 36-h fermentation periods (Table 2). This also demonstrated that fermentation is beneficial for improving the palatability and quality of FGCB.

3.8. Sensory evaluation of FGCB

The sensory scores of FGCB are presented in Fig. 4. In terms of color, odor, acidity, and rice scent, the L. plantarum fermented beverage received higher ratings than the unfermented sample. When compared with 12, 24, and 48 h of fermentation, fermentation for 36 h yielded beverages with the strongest rice aroma, typical lactic acid-dominated acidity, mild sweetness due to residual sugars, and the weakest beany flavor, resulting in optimal overall acceptability. In addition, L. plantarum fermentation was found to degrade the aldehydes in chickpeas and reduce the bitterness and beany flavor of the beverage, consistent with the results of volatile compound analysis.

Fig. 4.

Sensory evaluation of FGCB at different fermentation time points.

4. Conclusions

This study demonstrated that L. plantarum fermentation significantly improves the quality of FGCB. Following 36 h of fermentation, the TPC and TFC of FGCB increased, the antioxidant capacity was enhanced, and the α-amylase and α-glucosidase inhibition rates were elevated. Although simulated in vitro digestion slightly reduced the antioxidant and antidiabetic capacity of the beverage, the fermented groups consistently outperformed unfermented groups on these metrics, suggesting that L. plantarum fermentation enhances the stability of bioactive compounds during digestion and amplifies its health benefits. In addition, fermentation effectively reduced the undesirable odors of FGCB, and the unique flavor profile of the fermented beverage was shaped by elevated levels of substances such as 3-hydroxy-2-butanone, 1-hexanol, and 1-butanol. These findings indicate that FGCB, as a functional beverage, boasts enhanced sensory properties and is suitable for industrial production. Thus, it can be used to develop health products targeted at diabetes patients as well as healthy individuals. The characteristics of this beverage reflect its strong commercial potential. Moreover, the findings of this study demonstrate the promising role of fermented grain-based beverages in the functional food sector, facilitating further product development.

CRediT authorship contribution statement

Shuqi Wu: Writing – review & editing, Writing – original draft, Validation, Conceptualization. Bingjie Chen: Writing – review & editing, Writing – original draft, Validation. Xiao Wang: Supervision, Project administration, Data curation. Hongru Liu: Writing – original draft, Visualization, Software. Yi Zhang: Validation, Software, Investigation. Songheng Wu: Resources, Methodology, Investigation. Yongjin Qiao: Visualization, Investigation, Funding acquisition, Data curation. Xiaohui Li: Writing – review & editing, Methodology, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data that has been used is confidential.