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. 2026 Jul 30;38:104254. doi: 10.1016/j.fochx.2026.104254

Lactic acid bacteria fermentation drives metabolic remodeling to enhance anti-hyperuricemic potential and flavor quality of Portulaca oleracea juice

Xinhui Liu a,1, Yifan Zhang a,b,1, Yunlan Liu a, Huixian Wang a, Ruimin Wang a,⁎
PMCID: PMC13487313  PMID: 42621233

Abstract

Fermentation by lactic acid bacteria (LAB) has been shown to boost health benefits and enhance flavor. This study investigated the impact of Limosilactobacillus fermentum HNU022, Pediococcus acidilactici HNU323 and Limosilactobacillus fermentum HNU054 fermentation on Portulaca oleracea juice (POJ). The results revealed that fermentation boosted organic acids, antioxidant activities, and xanthine oxidase (XOD) inhibitory activity. Using non-targeted metabolomics, the study identified 823 differential metabolites, primarily concentrated in 5 pathways. Among them, isoflavonoid-related metabolites were upregulated, while purine metabolites were downregulated. The molecular docking results indicated that there were strong interactions between key metabolites and XOD‌. Electronic-tongue analysis showed improved flavor richness and reduced saltiness. In summary, LAB fermentation enhances the nutritional value, bioactivity, and flavor characteristics of POJ. Although molecular docking and in vitro activity assays indicate its antioxidant effects and XOD inhibitory capacity, these data still have certain limitations before in vivo activity studies are conducted for validation.

Keywords: Lactic acid bacteria fermentation, Portulaca oleracea juice, Metabolic profile, Molecular docking, Electronic tongue

1. Introduction

Portulaca oleracea L., also named purslane, belonging to the family Portulacacea, is an annual succulent herb (Hou et al., 2021; Li, Ren, Yang, Lu, & Tan, 2024). It is widely consumed as a leafy vegetable and traditional food ingredient across temperate and tropical regions throughout the world. Portulaca oleracea is commonly consumed as a functional food ingredient or dietary supplement due to its rich content of phenolic acids, flavonoids, alkaloids, and polysaccharides (Obukohwo, 2024). The Portulaca oleracea contains multiple functional features, such as anti-inflammatory, anti-hyperglycemia, anti-trioxypurine, anti-oxidation, neuroprotection, and immune regulation (Cannavacciuolo et al., 2025). Portulaca oleracea is food and medicine share the same origin and has been designated a “Global Panacea”, indicating its considerable potential as human food. Portulaca oleracea has the potential to surpass wild vegetables and dietary supplements in traditional medicine (Jalali & Ghasemzadeh Rahbardar, 2022). Despite the well-established extensively demonstrated of Portulaca oleracea's nutritional benefits, research on the bioactivity of its fermented products remains limited.

Fermentation is one of the oldest food biological processing methods, involving the role of microorganisms such as bacteria and fungi. Lactic acid bacteria (LAB) fermentation is one of the most suitable tools for developing the functional potential of plant matrix. It improves the bioavailability and biological activity of phytochemicals, and enriches the plant matrix with functional bacterial metabolites (Leonard, Zhang, Ying, Adhikari, & Fang, 2021). Recent researches have showed that LAB fermentation of herb juice increases antioxidant activity, bioactive substance content, and biological activity. This process also induces metabolomic changes to improve the herbs juice's sensory and flavor characteristics. (Chai et al., 2024; Wang et al., 2025). Prior investigations have demonstrated LAB fermentation and their fermentation broth can reduce serum uric acid and xanthine oxidase (XOD) activity while regulating metabolic pathways related to purine metabolism, amino acid metabolism, the tricarboxylic acid cycle, the urea cycle, tryptophan-derived metabolites, intestinal urate excretion, along with an assessment of fermentation safety (Rao et al., 2024; Dong et al., 2025). Therefore, the fermentation of Portulaca oleracea juice (POJ) as a positive human health nutrition fermented product supplement is of great significance.

This study aims to systematically elucidate the bioactive potential, differential metabolites, and sensory characteristics of probiotic-fermented POJ, as well as investigate in vitro inhibitory activity against XOD. In this study, fermented POJ produced by probiotics Limosilactobacillus fermentum HNU022 (L. fermentum HNU022), Pediococcus acidilactici HNU323 (P. acidilactici HNU323) and Limosilactobacillus fermentum HNU054 (L. fermentum HNU054) was employed to determine XOD inhibitory activity and antioxidant activity as well as organic acid content, total phenolic content (TPC) and total triterpene content (TTC). Furthermore, differential metabolites, which is identified through non-targeted metabolomic analysis, was employed to evaluate the binding energy of XOD by molecular docking technology, and verify their potential hypouricemic activity in vitro. Key metabolite levels were further subjected to targeted analysis and validation using standards. Simultaneously, sensory characteristics of fermented POJ were evaluated by electronic tongue (E-tongue). In conclusion, this study aimed to establish a link between fermentation-driven metabolic changes and urate-lowering activity, thereby providing both theoretical insight and practical data support for the development of LAB-fermented POJ as a functional food ingredient or beverage.

2. Materials and methods

2.1. Plant materials and reagents

Portulaca oleracea was collected from Wuzhishan, Hainan, China. LAB strains, including L. fermentum HNU022, P. acidilactici HNU323 and L. fermentum HNU054 are obtained from tropical health food biological manufacturing team of Hainan University. The MRS medium was purchased from Shanghai Maclin Biochemical Technology Co., Ltd. Aladdin Biochemical Technology provided DPPH (1,1-Diphenyl-2-picrylhydrazyl), Trolox, XOD and xanthine. Nanjing Herb Source Bio-Technology Co., Ltd. supplied the organic acids and phenols standards.

2.2. Preparation of POJ

Fresh Portulaca oleracea was homogenized with distilled water at a ratio of 1:0.5 (w/v). An equal volume of water was then added, and ultrasonic-assisted extraction was performed at 55 °C and 200 W for 1.5 h. After cooling, the mixture was centrifuged at 6000 rpm for 5 min, and the supernatant was collected. Subsequently, 6% prebiotics (galactooligosaccharide: fructooligosaccharide = 1:4) were added and dissolved, followed by sterilization at 121 °C for 15 min to obtain sterile POJ.

2.3. LAB strains and POJ fermentation

Twenty candidate LAB strains were screened using the XOD in vitro inhibitory activities of their intracellular and extracellular metabolites. L. fermentum HNU022, L. fermentum HNU054, and P. acidilactici HNU323 were selected because their extracellular metabolites produced the three highest inhibition rates (60.96%, 46.55%, and 37.84%, respectively; Table S1).

The strains L. fermentum HNU022 (HNU022), P. acidilactici HNU323 (HNU323), and L. fermentum HNU054 (HNU054) were activated by culturing in MRS liquid medium to achieve exponential-phase growth. The supernatant was removed after centrifuging the activated cultures at 4000 ×g for 5 min. Then redissolved in an equal amount of 0.9% sterile saline to prepare a uniform suspension. This resultant cell suspension was inoculated at 2% (v/v) into the POJ and fermented at 37 °C for 36 h to yield the fermented POJ (Wang et al., 2025). For each LAB strain, three independent fermentation replicates were carried out.

2.4. LAB viability count and physicochemical property analysis

The viable counts of LAB in the fermented POJ were quantified by the plate count method and measured in lg CFU/mL for units. The pH level is determined with the American Ohmstad ST300 series portable pH meter, and total soluble solids (TSS) contents were measured with a digital refractometer and reported as °Brix. Each measurement was performed on three independent biological replicates.

2.5. Organic acids

The organic acid contents were assessed using a method adapted from a prior study (Zhang et al., 2023). In brief, after centrifugation at 4000 rpm and 4 °C for 5 min, the supernatant was filtered through a 0.22 μm filter membrane. Analysis was performed on an Agilent 1260 Infinity II HPLC system equipped with a VWD detector and an Agilent ZORBAX SB-Aq column (4.6 × 250 mm, 5 μm). The column temperature was 40 °C. Isocratic elution used perchloric acid (pH 2.5) at 0.6 mL/min, with detection at 210 nm, injection volume 10 μL, and run time 20 min, expressed as mg/mL. Three independent fermentation batches were analyzed, each in duplicate injection.

2.6. TPC and TTC analysis

TPC was determined using the Folin-Ciocalteu method (Wang et al., 2022). Briefly, 100 μL of gallic acid standard or sample solution was combined with 50 μL of Folin-Ciocalteu reagent and left to react in darkness for 5 min. Then, 300 μL of 20% sodium carbonate solution was added, followed by another 8 min incubation in the dark. A 200 μL portion of the mixture was moved to a 96-well plate, and absorbance was measured at 765 nm. TPC was represented as gallic acid equivalent (GAE) in μg GAE/mL.

According to the method (Zhang et al., 2023), the 50 μL LAB-fermented POJ or different concentrations of ursolic acid standard solution was transferred into brown EP tubes andevaporated in a 90 °C water bath. After undergoing acid hydrolysis, the absorbance of a 200 μL sample of the resulting solution was measured at 548 nm in a microplate, using methanol as the blank. The TTC content was calculated as ursolic acid equivalent and expressed as mg/g FW (fresh weight). Each measurement was conducted in triplicate for every strain, and the results were averaged.

2.7. Analysis of antioxidant activity

For FRAP assay, the 50 μL of sample was mixed with 450 μL of FRAP working solution containing 300 mM acetate buffer, 10 mM TPTZ, and 20 mM FeCl3·6H2O (10:1:1, v/v/v). The mixture was incubated at 37 °C for 30 min in the dark, and absorbance was read at 593 nm. The CUPRAC assay was performed by reacting 50 μL of sample with 450 μL of a solution composed of 10 mM CuCl2, 7.5 mM neocuproine, and 1 M NH4Ac buffer (1:1:1, v/v/v). After 30 min of incubation at 25 °C, absorbance was measured at 450 nm (Szydłowska-Czerniak, Kowaluk, Strzelec, Sawicki, & Tańska, 2025; Zheng et al., 2023).

The DPPH radical scavenging activity was determined by mixing 50 μL of sample with 400 μL of 100 μM DPPH-ethanol solution, followed by 30 min dark incubation at room temperature. Absorbance was then measured at 517 nm (Wang et al., 2022). All results were expressed as Trolox equivalents in μmol TE/100 mL, and each assay was performed in triplicate.

2.8. XOD inhibition activity evaluation

An enhanced version of the former XOD inhibition activity assay was employed (Dong et al., 2025; Wang et al., 2026; Yang et al., 2025). Briefly, a control solution was prepared by combining XOD (0.2 U/mL) with PBS buffer while the test sample was prepared by adding 50 μL of the sample to the same XOD/PBS mixture. The resulting mixtures were initially incubated for 10 min at 37 °C. Following this, 150 μL of a 0.2 mM xanthine solution was added, and a further 10 min incubation was carried out at the same temperature. The absorbance was measured at 295 nm. All measurements were performed in triplicate.

2.9. Non-targeted metabolomic analysis

The metabolic characteristics of POJ subjected to LAB fermentation were investigated by non-targeted metabolomics, based on a previously reported protocol (Meng et al., 2022; Wang, Bai, et al., 2025). In summary, 100 μL of fermented POJ was combined with 400 μL of methanol: water (4:1, v/v) containing 0.02 mg/mL 2-chloro-L-phenylalanine as internal standard. The resulting mixture was vortexed and then underwent ultrasonic-assisted extraction for 30 min at 5 °C. To facilitate protein precipitation, it was subsequently stored at -20 °C for an additional 30 min and centrifugated at 15,000 ×g for 20 min to obtain supernatant for UHPLC analysis. Metabolite separation and detection were performed on an Ultimate 3000 UHPLC system coupled to a Q-Exactive HF-X mass spectrometer (Thermo Scientific, USA). Using an electrospray ionization (ESI) source, data were acquired in positive and negative ionization modes, respectively. Each measurements were performed in triplicate, and the results were averaged. Metabolite identification was performed by matching the accurate m/z and retention times against the Human Metabolome Database (HMDB). Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was employed for functional annotation and metabolic pathways analysis based on differential metabolites (Chen et al., 2026; Tai et al., 2026). Enrichment significance for pathways was established at p < 0.05.

2.10. Molecular docking

The molecular docking of selected metabolites with XOD was carried out employing AutoDock (v. 4.2). Ligands' 3D structures were acquired from the PubChem database by utilizing their CAS. The preparation of the ligands and receptor involved tasks such as adding hydrogen atoms, computing charges, and defining atom types in AutoDock Tools. Visualization of the docking results was done in 3D with PyMOL and in 2D with Discovery Studio 2019 (Girawale, Meena, Yadav, & Kodam, 2023). The strength of the interaction between the ligands and XOD was evaluated using binding affinity (kcal/mol).

2.11. Qualitative and quantitative analysis of key metabolites

The previous experimental method was referenced and optimized (Qin, Luo, Qiu, Zhang, & Yang, 2024), conducting qualitative and quantitative analysis under the following experimental conditions. The 200 μL POJ and fermented POJ sample was transferred into a 1.5 mL centrifuge tube and centrifuged at 12,000 ×g and 4 °C for 10 min. The supernatant was collected and filtered through a 0.22 μm membrane and analyzed for targeted qualitative and quantitative analysis using an Agilent 1290 Infinity III-6495D mass spectrometer (Agilent Technologies, USA). Chromatographic separation was performed on a C18 column (100 mm × 2.1 mm i.d., 1.8 μm) with 3 μL injection volume, 40 °C column temperature and 0.3 mL/min velocity. A linear gradient elution was applied using mobile phase A (95% water/5% acetonitrile with 0.1% formic acid) and mobile phase B (acetonitrile): 0–1.5 min, 0%–5% B; 1.5–2 min, 5%–10% B; 2–4.5 min, 10%–30% B; 4.5–5 min, 30%–100% B; 5–6.3 min, 100% B; 6.3–6.4 min, 100%–0% B, and 6.4–8 min 0% B. The sample was then subjected to electrospray ionization, and mass spectrometry signals were acquired using both positive and negative ion scanning modes. Mass spectrometric parameters were set as follows: scan type 70–1050 m/z, ion source heater temperature 350 °C, and spray voltages of +3400 V and − 3400 V. Standard solutions were prepared to establish calibration curves for the qualitative and quantitative determination of target analytes.

2.12. Electronic tongue assessment

The taste profile of sample was examined using an E-tongue, as per the outlined method described by (Li, Ren, et al., 2024). Prior to measurement, the E-tongue system was started up, calibrated, and rinsed following standard procedures. The E-tongue system incorporates seven distinct taste sensors, namely sourness (AHS), complex taste-A (PKS), saltiness (CTS), umami (NMS), complex taste-B (CPS), sweetness (ANS), and bitterness (SCS), to discern a variety of taste characteristics. All measurements were performed at 25 °C using an AgCl reference electrode. Sensory data were collected automatically through an integrated software platform to ensure accuracy and reproducibility.

2.13. Statistical analysis

All experiments were performed in triplicate, and the data are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 26.0 (IBM, USA). One-way analysis of variance (ANOVA) followed by Duncan's multiple range test was used to determine significant differences among groups. A probability value of p < 0.05 was considered statistically significant. Graphs were generated using GraphPad Prism 9.0.

3. Result and discussion

3.1. Shifts in the physicochemical aspects of POJ

3.1.1. Viable cell counts variations

Viable cell counts serve as a pivotal composite metric for gauging LAB fermentation performance, metabolic productivity, and substrate adaptability. Upon 36 h of fermentation, the total number of viable cells in POJ fermentation significantly increase (p < 0.05), as shown in Fig. 1A. Among the tested strains, fermentation with P. acidilactici HNU323 yielded the highest bacterial count in POJ at 8.86 lg CFU/mL, whereas L. fermentum HNU022 and L. fermentum HNU054 produced 8.39 and 8.36 lg CFU/mL. All strains exceeded 8 lg CFU/mL, a threshold associated with health benefits (Li et al., 2024), confirming that POJ is a suitable substrate for LAB growth.

Fig. 1.

Fig. 1

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Physicochemical property variations associated with LAB fermentation: viable cell count (lg CFU/mL) (A), pH (B), total soluble solids (°Brix) (C), and organic acid contents (mg/mL) (D) of POJ and fermented POJ. Columns with different lowercase letters are significantly different (p < 0.05, one-way ANOVA followed by Duncan's test).

3.1.2. Changes in pH and TSS

The pH value is a critical parameter for measuring the acidity of fermented products, playing a decisive role in flavor development and quality control. As shown in Fig. 1B, the pH of POJ significantly decreased through fermentation with the three bacterial strains (p < 0.05), compared to the unfermented control (4.76 ± 0.09). The lowest pH was recorded in POJ fermented by HNU323 (3.63 ± 0.02), followed by HNU022 and HNU054, with pH values of 3.64 ± 0.01 and 3.72 ± 0.02, primarily due to LAB metabolism of sugars into organic acids (Alan, 2024). Meanwhile, TSS showed no significant difference before and after fermentation (Fig. 1C). This may be explained by LAB-promoted protein degradation increasing water-soluble proteins, which offsets the consumption of soluble sugars (An et al., 2022).

3.1.3. Organic acid profiles of fermented POJ

LAB fermentation produces various organic acids, as presented in Fig. 1D. Compared to other organic acids, the malic acid content in the fermented POJ was significantly elevated, followed by tartaric, lactic, succinic, citric, and oxalic acids. Malic acid was the predominant organic acid in fermented POJ, with the highest level observed in the HNU022 group, followed by HNU323 and HNU054. Compared with the POJ group, fermented samples showed increased levels of tartaric acid and lactic acid, with lactic acid exhibiting a strain-dependent accumulation pattern similar to that of malic acid. Although present at relatively low concentrations, tartaric acid and lactic acid are important contributors to flavor development (Rabah, Rosa do Carmo, & Jan, 2017; Ruan et al., 2026). The changes in malic acid and lactic acid suggest active organic acid conversion during fermentation, potentially involving malolactic fermentation and tricarboxylic acid (TCA) cycle-related metabolism (Duan et al., 2023). Consistent with previous studies, lactic acid production varied among LAB strains (Punia Bangar, Suri, Trif, & Ozogul, 2022). Succinic acid and citric acid, two key TCA cycle intermediates, were also detected. Compared with POJ, succinic acid increased after fermentation, whereas citric acid remained at lower levels, reflecting strain-specific differences in organic acid metabolism (Yang et al., 2023).

Oxalate is a key concern for kidney stone patients. In this study, oxalic acids was not detected in HNU022 and HNU054 group. The oxalic acids content in the HNU323 (166.85 ± 4.66 μg/mL) was significantly lower than POJ (486.72 ± 4.47 μg/mL), and also below the threshold for high-oxalate sample classification (Gaur & Kumar, 2025). These results demonstrate that the oxalate content in some strains was not completely eliminated after fermentation, and the dynamic changes in the proportion of soluble oxalate to total oxalate remain unknown. Moreover, systematic evaluations of safe intake levels for high-risk populations such as individuals susceptible to kidney stones have not been performed. Thus, the food safety of fermented purslane still is needed to evaluate.

Overall, LAB fermentation promoted organic acid transformation, contributing to the flavor complexity and functionality of fermented POJ.

3.2. The concentration of bioactive substances and the biological properties of fermented POJ

3.2.1. Changes in the TPC and TTC

The phenolic compounds correlate with multiple biological activities. As shown in Fig. 2A, the results indicated that TPC of POJ fermented with HNU323 was significantly increased than the unfermented POJ and the samples fermented with strains HNU022 and HNU054. In this study, the observed increase in TPC of fermented POJ may be attributed to a corresponding increase in glycosidases and phenol-esterases, which facilitate the hydrolysis of bound phenolic compounds into free phenolics (Meng, Wang, Hao, Zhu, & Wang, 2023). The results showed that LAB fermentation promotes the release of polyphenols in POJ.

Fig. 2.

Fig. 2

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Variations in bioactive substances and bioactive activities. Total phenolic content (TPC) (A), total triterpene content (TTC) (B), xanthine oxidase inhibition (C), FRAP (D), CUPRAC (E), DPPH (F).

Triterpenoids are key bioactive compounds in Portulaca oleracea, acting alongside flavonoids, alkaloids, and sterols to confer anti-hyperuricemic, anti-inflammatory, antioxidant, antitumor, and neuroprotective activities (Zhang et al., 2021). As illustrated in Fig. 2B, the TTC was substantially enriched in fermented POJ, demonstrating significantly changes (p < 0.05) compared with unfermented POJ. Notably, HNU323-fermented POJ exhibited the highest triterpenoid concentration (5.99 ± 0.03 mg/g FW), followed by HNU054 (5.60 ± 0.21 mg/g FW) and HNU022 (3.38 ± 0.34 mg/g FW). The result may be attributed to the abundant nutrient availability during the early stages of fermentation, which promotes triterpenoid biosynthesis (Wang et al., 2024). Consistent with our findings, comparable increases in TTC following LAB fermentation have been found in Lessertia frutescens and sun-dried Lithocarpus tea (Gao, Yang, Liu, & Wang, 2025; Lou, Mu, Liu, Xun, & Hu, 2023).

3.2.2. Fermented POJ'S XOD inhibition and antioxidant capabilities

XOD is a key enzyme regulating uric acid production. Inhibiting of XOD activity has been recognized as an effective strategy to reduce uric acid levels, thereby alleviating symptoms associated with hyperuricemia and related conditions such as gout (Zhang, Zhu, Gu, Feng, & Gao, 2024). The study indicates that fermented POJ significantly inhibited XOD activity compared to the unfermented POJ (p < 0.05, Fig. 2C). Notably, the HNU022 and HNU054 groups exhibited more pronounced inhibition (85.93 ± 4.56% and 84.63 ± 3.58%, respectively), while the HNU323 group reached 69.91 ± 0.99%, which was considerably higher than unfermented POJ (15.37 ± 1.35%). Recent research suggests that the enhanced XOD inhibition may be closely associated with increased levels of polyphenols and triterpenoids in the fermented sample (Ma et al., 2026; Mehmood et al., 2019). In summary, LAB-fermented POJ can reduce uric acid production in vitro by inhibiting XOD activity, indicating that fermentation significantly enhances its ability to suppress uric acid generation in vitro.

This study employed three detection methods, including FRAP, CUPRAC, and DPPH radical scavenging activity, to comprehensively evaluate the antioxidant capacity of POJ. As shown in Fig. 2D-E, all fermented POJ groups had higher FRAP values than unfermented POJ, with no significant difference among three fermentation treatments. HNU054 demonstrating the highest level (106.37 ± 1.48 μmol TE/100 mL), followed by HNU323 (106.06 ± 1.01 μmol TE/100 mL) and HNU022 (105.72 ± 2.81 μmol TE/100 mL). Meanwhile, the trend of CUPRAC results in each group after fermentation is consistent with the FRAP value. The HNU323 group exhibited the highest CUPRAC value (162.99 ± 2.61 μmol TE/100 mL), followed by HNU054 group (158.35 ± 1.33 μmol TE/100 mL) and HNU022 group (158.10 ± 3.61 μmol TE/100 mL). These increases in FRAP and CUPRAC values indicate an enhanced reducing capacity, which was positively correlated with the concentrations of bioactive components, particularly polyphenols and triterpenoids. As reported previously, augmented FRAP and CUPRAC values in goji berry and pitaya juices are principally mediated by the accumulation of phenolic and flavonoid compounds during LAB fermentation (Sakda et al., 2025). Moreover, the fermentation process aids in the breakdown of cell structures, potentially enhancing antioxidant activity by releasing various antioxidant compounds (Shi, Wang, & Li, 2023).

A distinct pattern was observed for DPPH radical scavenging activity. As shown in Fig. 2F, the DPPH radical scavenging activity of POJ was not significantly affected (p > 0.05) by 36 h of LAB fermentation. This result contrasts with the marked increases observed in FRAP and CUPRAC values. Accordingly, it may be inferred that, under the current experimental conditions, fermentation contributed to an enhancement in reducing capacity while exerting no significant influence on DPPH radical-scavenging activity. Our results align with previous studies indicating that lactic acid produced during LAB fermentation acidifies POJ, which may attenuate DPPH scavenging capacity (Li et al., 2026). Earlier research also reported no notable difference in DPPH radical scavenging activity between LAB-fermented and unfermented cauliflower stems (Zhang et al., 2024). These observations suggest that DPPH radical scavenging activity varies with LAB strains and fermentation substrates.

Overall, LAB generally increased the FRAP/CUPRAC and reduced the in vitro XOD inhibitory activity of POJ, but it did not affect the DPPH radical-scavenging activity.

3.3. Metabolic profile analysis of fermented POJ

The non-targeted metabolomics was employed to putatively annotate the metabolites of POJ. A total of 1398 metabolites were putatively annotated in positive ion mode and 1432 in negative ion mode. Among the 2830 putative annotations, 1577 were classified as B(i) and 1253 as B(ii). Furthermore, following comparison against self-built standard libraries, Metlin, and LipidBlast, 65.7% of metabolites in positive ion mode exceeded the platform detection threshold (Fragmentation Score > 35), compared with 46.1% in negative ion mode. The confidence information of metabolites is shown in Table S2, and the total-ion chromatograms are provided in the supplementary Fig. S1-S2. And, PLS-DA (Partial least squares Discriminant Analysis) revealed substantial inter-group metabolic differences between the unfermented and fermented POJ groups (Fig. 3A-B), which indicating that the metabolites of POJ underwent significant changes due to fermentation. Meanwhile, PLS-DA of metabolites among different fermentation groups also demonstrated distinct inter-group differences. Notably, metabolite profiles differed obviously among fermentation groups with the selection of different fermentation strains. The HNU323 strain exhibits particularly pronounced differences in both negative and positive ion modes compared to other groups. The Venn diagram also showed metabolic differences among all groups (Fig. 3C). KEGG database pathway analysis for all recognized metabolites revealed enriched metabolic pathways including metabolism (92.66%), environmental information processing (4.98%), genetic information processing (1.62%), cellular process pathways (0.62%), and biological system pathways (0.12%, Fig. 3D). Through comparative analysis in the HMDB database, the identified compounds were assigned to 16 categories. Among them, four predominant categories (>10.00%) were organic acids and derivatives (25.00%), lipids and lipid analogues (21.70%), organic heterocyclic compounds (14.77%), and organic oxides (13.96%), as shown in Fig. 3E.

Fig. 3.

Fig. 3

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Non-target metabolomic analysis. PLS-DA score plots derived from positive (A) and negative (B) ion mode, histogram of KEGG (D), and metabolites classification of HMDB (E), heatmap displaying the relative abundance of the top 30 differential metabolites in POJ (F), KEGG-based topology enrichment of the 20 most significant differential metabolites in POJ (G).

Employing the criteria of VIP > 1 and p < 0.05, the 823 metabolites were ultimately identified as differential metabolites in this study. Metabolites with relative abundances in the top 30 were selected for metabolite heatmap analysis between the control group and the fermentation group (Fig. 3F). Compared with unfermented POJ, fermented POJ showed significantly increased abundances of flavonoids and phenylalanine metabolites, including DL-leucic acid, sedoheptulose, cyclokievitone, glyceofuran, 4-hydroxyphenyllactic acid, recoflavone, 3,4′-dihydroxypropiophenone, D-3-phenyllactic acid, phenethyl rutinoside, valtrate, homofuraneol, hypoxanthine, and dihydrocoumarin. As a crucial amino acid for humans, DL-Leucine is part of the branched-chain amino acids, plays a critical role in protein synthesis and multiple metabolic pathways (Li et al., 2024). Recent studies have revealed that recoflavone has completed phase III clinical trials in South Korea for gastritis treatment, demonstrating its potential in reducing inflammation (Butler, Robertson, & Cooper, 2014). Meanwhile, the abundances of most nucleotide metabolites in fermented POJ showed significant downregulation, such as guanine, inosine, xanthosine, adenosine, and guanosine. Notably, the level of hypoxanthine, a key precursor for uric acid synthesis within the purine metabolic pathway, was markedly increased. This phenomenon may result from altered levels of specific metabolites interfering with hypoxanthine metabolism, but the potential mechanism requires further investigation.

Meanwhile, the KEGG pathway analysis was conducted on the metabolic set composed of 823 metabolites. According to the relative importance scores, enrichment and functional annotation were conducted on the top twenty metabolic pathways. Among these, the five highest-ranked pathways were glycine, serine and threonine metabolism, flavone and flavanol biosynthesis, histidine metabolism, nucleotide metabolism, and the TCA cycle (Fig. 3G), which were closely related to the enrichment or inhibition of differential metabolites (Fig. 3D). In summary, topological analysis indicated that the metabolic profile of LAB-fermented POJ differs from that of unfermented POJ, potentially promoting the relevant biotransformation of POJ metabolites.

3.4. Functional pathway analysis of key metabolic intermediates

According to the metabolomic cluster analysis heatmap, KEGG topological pathway analysis results, and the fundamental chemical properties of the key metabolites in Portulaca oleracea, we constructed the functional metabolic pathway associated with these key metabolites (Fig. 4). In the metabolite clustering analysis heatmaps of the unfermented and fermented groups, metabolites involved in “nucleotide metabolism”, “TCA cycle”, and “flavonoid and flavanol metabolism” showed significant changes before and after fermentation. As shown by the trend of key metabolite changes in the nucleotide metabolism pathway, almost all nucleotide metabolites, including guanosine, inosine, adenosine, xanthosine, and guanine, exhibited significantly downregulated expression levels after fermentation. Similar studies have shown that the fermentation of spontaneous fermentation of tomatoes also reflect a decrease in the content of nucleotide metabolites such as guanosine, guanine, and adenosine (Song, Zhou, Li, Huang, & Zhou, 2024). The TCA cycle is the main catabolic process for carbohydrate metabolism, fatty acid metabolism, and amino acid metabolism in organisms (Choi, Son, & Baek, 2021). Succinate and malate, as important intermediate metabolites in the TCA cycle, showed decreased expression levels after fermentation. In the TCA cycle pathway, glycine, serine, and cysteine can be converted to pyruvate via amino acid transamination and decarboxylation. Meanwhile, methionine and propionic acid are transformed into succinyl-CoA, and histidine is converted to α-ketoglutaric acid (Wang et al., 2021). All of these are important intermediate metabolites in the TCA cycle, and the levels exhibited variations across the all groups in this study.

Fig. 4.

Fig. 4

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The functional metabolic pathway associated with fermented POJ key metabolites.

Meanwhile, within the flavonoe and flavanol biosynthesis pathways, phenylalanine undergoes transamination to form p-hydroxyphenylalanine, which is subsequently converted into isoflavones for further catabolism (Mao, Luo, & Cai, 2025). We noticed that fermentation significantly upregulated the content of all phenylalanine metabolites, with the exception of tyrosine, including tyrosine, 4-phenylacetic acid, and D-3-phenylalanine. Tyrosine can be converted into fumarate to participate in the TCA cycle for further catabolism (Wang et al., 2023). The significant increase in phenethyl rutinoside content observed in the fermentation group may be attributed to its formation via the reaction between 4-phenylacetic acid and rutin-like substances in POJ. Phenethyl rutinoside has been shown in existing research literature to exhibit biological activities such as potent antioxidant capacity (Mashiane et al., 2021). As a key phenylalanine metabolite, trans-cinnamic acid can be metabolized to succinate for entry into the TCA cycle or converted into dihydrocoumarin, thereby exhibiting distinct functional activities (Gu, Li, & Zhou, 2023). Trans-cinnamic acid content was also significantly upregulated in the fermentation group. Previous research has indicated that trans-cinnamic acid exhibits notable antioxidant and lipid-lowering properties (Nouni, Theodosis-Nobelos, & Rekka, 2023). The contents of cyclokievitone, glyceofuran, and recoflavone in fermented POJ, which are transformed from isoflavones, were all significantly upregulated in the fermentation group. Previous research showed that isoflavones can ameliorate hyperuricemia by inhibiting XOD activity to reduce uric acid production, while simultaneously leveraging their free radical scavenging capacity to alleviate renal oxidative stress induced by hyperuricemia (Duan, Qi, Liu, Zhang, & Liu, 2022).

It is noteworthy that in the nucleotide metabolism pathway of the fermentation product, all purine metabolites exhibited a significant downward trend except hypoxanthine. Xanthine is primarily metabolized into uric acid by the XOD and then excreted in the form of urine (Yang et al., 2024). However, hypoxanthine, as the precursor of xanthine, showed a notable upward trend in its content, indicating that the pathway from xanthine to uric acid is severely inhibited. Conversely, the contents of flavonoe and flavanol in the fermented portulaca metabolites all exhibited an upward trend. Comprehensive analysis of the above results indicates that fermented portulaca may effectively inhibit XOD activity via the metabolism of flavonoids and flavanols.

3.5. Molecular docking results

Based on the above findings, molecular docking was performed between XOD and the key differential metabolites of fermented POJ to analyze their specific binding sites and binding energies, thereby elucidating the inhibitory effects of these differential metabolites on XOD activity. XOD served as the receptor after dehydration and ligand removal by hydrogenation treatment. Sixteen enriched metabolites were subjected to hydrogenation protonation as ligands for molecular docking. The results revealed that among the 16 enriched metabolites docked, 9 exhibited binding energies of less than -5 kcal/mol, as shown in Fig. 5J. The lowest binding energy was observed for recoflavone (-9.54 kcal/mol), while the highest was for succinic acid (-2.86 kcal/mol). A lower binding energy corresponds to increased stability in the interaction with the XOD enzyme receptor (Wee, Loh, Lam, & Ismail, 2023). Notably, the top four metabolites with the lowest binding energies were all isoflavones, indicating that isoflavones likely contribute significantly to the hypouricemic effects observed in fermented POJ metabolites. Furthermore, the hydrogen bonds and amino acid residues for the top nine metabolites (binding energy < -5 kcal/mol) were analyzed and visualized in three dimensions using the PyMOL software. The 2D structures, as well as non-covalent interactions such as pi-pi bonds, pi-cation bonds, and pi-Alkyl bonds, between the molecular dockings were analyzed using Discovery Studio software, as illustrated in Fig. 5 A-I.

Fig. 5.

Fig. 5

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Molecular docking model of Recoflavone (A), Cyclokievitone (B), Glyceofuran (C), Phenethyl Rutinoside (D), Dihydrocoumarin (E), 7-oxo-11- Dodecenoic Acid (F), Valtrate (G), Histamine (H), L-Malic Acid (I) and XOD. The binding energy of 16 fermented POJ metabolites with XOD (J). Key metabolites IC50 on Xanthine oxidase in vitro (K). Qualitative and quantitative analysis of key metabolites (L).

The binding energy between recoflavone and XOD was the lowest binding energy, and formed eight hydrogen bonds and interacted with nine amino acid residues in the XOD active site. The 2D structure diagram also reveals one pi-cation bond and two pi-Alkyl bonds with the XOD enzyme, indicating high XOD enzyme inhibitory activity of recoflavone. Although dihydrocoumarin does not belong to the isoflavone class of metabolites, its binding energy with XOD also reaches -6.76 kcal/mol. The binding analysis revealed that dihydrocoumarin interacts with XOD via three hydrogen bonds involving two residues. The THR-1011 residue engages in a dual hydrogen-bonding interaction, which enhances the stability of the complex. Although dihydrocoumarin forms only three hydrogen bonds with XOD, analysis of its molecular docking 2D diagram reveals additional stabilizing interactions. Specifically, it forms five pi-alkyl bonds with ALA, LEU, and PHE residues, and one pi-sigma bond with a PHE residue. The results indicated that the cumulative effect of these weaker non-bonding interactions was also significant, collectively contributing to the stable binding of dihydrocoumarin to XOD. Dihydrocoumarin acts as a competitive or non-competitive inhibitor of XOD. It inhibits the enzyme by blocking its active site, preventing the oxidation of xanthine into uric acid (Antoniolli, de Moraes, da Costa, de Campos, & Coelho, 2025; Lin et al., 2008). Concurrently, we detected several organic acids metabolites with relatively low molecular docking binding energies during the fermentation of POJ, such as gulonic acid and D-3-phenyllactic acid. Gulonic acid is converted into ascorbic acid, thereby reducing oxidative stress, and may exert a uric acid-lowering effect by inhibiting XOD activity (Gad & Sirko, 2024). D-3-phenyllactic acid boosts the antioxidant properties of food and influences gut microbiota, thereby improving overall metabolic health (Kim et al., 2024; Zhang et al., 2024). In summary, isoflavone-related metabolites and several organic acids may contribute to XOD inhibition. Accordingly, we further obtained the corresponding standards and performed subsequent in vitro validation experiments for XOD inhibitory activity.

To validate the XOD inhibitory potential predicted by molecular docking, we performed in vitro XOD inhibition assays on six compounds exhibiting binding energies < -5 kcal/mol, and the results are shown in Fig. 5K. The results indicated that Recoflavone exhibited the strongest XOD inhibitory activity (IC50 = 14.625 μmol/L), followed by Valtrate (IC50 = 27.04 μmol/L) and Dihydrocoumarin (IC50 = 79.893 μmol/L). Despite the above experiments confirm the in vitro XOD inhibitory potential of these test compounds, in vivo uric acid-lowering effects need to be verified in the future.

To further validate the identification reliability, qualitative and quantitative analysis of the 6 key metabolites in each group were verified using their corresponding standards. Standard curve information for the six key metabolites is detailed in Table S3. As shown in Fig. 5L, the results indicate that all six compounds were detected and confirmed across all groups, and their concentration trends were consistent with those observed in untargeted metabolomics analysis. These findings can support the reliability and accuracy of metabolite identification.

3.6. E-tongue results

The electronic tongue provides an efficient and unbiased approach to evaluate the sensory attributes of fermented juices by quantifying their fundamental taste profiles. As shown in Fig. 6, LAB fermentation markedly altered the taste profile of POJ. The principal component analysis (PCA) was performed comparing the unfermented control group with the three fermented experimental groups and revealed that the four groups clustered into four distinct regions, with PC1 and PC2 together contribute to 97.359% of the cumulative variance (Fig. 6A).

Fig. 6.

Fig. 6

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PCA score plots (A) and E-tongue radar chart (B) of four groups based on E-tongue detection. Taste dimensions encompass sourness(AHS), complex taste-A (PKS), saltiness (CTS), umami (NMS), complex taste-B (CPS), sweetness (ANS), and bitterness (SCS).

As shown in Fig. 6B, the most notable changes were observed in the HNU022 and HNU054 groups, where umami increased markedly and saltiness decreased substantially. Bitterness and sourness also increased in these two groups, while sweetness showed a moderate reduction. For the HNU323 group, only slight decreases in saltiness, bitterness, and sweetness were observed. The marked increase in umami across all fermented samples, particularly in HNU022 and HNU054. This is consistent with previous reports that LAB fermentation produces umami compounds such as succinic acid and lactic acid, and that disodium succinate synergizes with glutamic acid to enhance umami (Tong, Li, Liu, & Zheng, 2025; Wu, Blank, Zhang, & Liu, 2022). Overall, LAB fermentation enriches the flavor profile of POJ, with strain-dependent effects.

4. Conclusion

This study demonstrates that LAB fermentation effectively enhances the functional profile of POJ. Fermentation significantly increased the contents of key bioactive components, including TPC and TTC, thereby boosting its in vitro antioxidant capacity and XOD inhibitory activity. Non-targeted metabolomics revealed a comprehensive reshaping of the metabolic landscape in POJ, characterized by the marked upregulation of beneficial flavonoid (particularly isoflavones) and the downregulation of most purine metabolites. Molecular docking further identified specific metabolites, such as recoflavone, as potent XOD inhibitors, and in vitro IC50 assays measuring their XOD activity revealed the potential mechanism underlying their uric acid-lowering effects. Concurrently, E-tongue analysis confirmed that fermentation optimizes the sensory attributes of POJ. Collectively, these findings indicate that POJ fermented with LAB exhibits in vitro inhibition of XOD and antioxidant activity. Nevertheless, further in vivo studies are needed to confirm its uric acid-lowering effects and elucidate the underlying molecular mechanisms. These investigations will provide a solid scientific basis for the development of functional foods.

CRediT authorship contribution statement

Xinhui Liu: Writing – original draft, Methodology, Conceptualization. Yifan Zhang: Writing – review & editing, Software, Data curation. Yunlan Liu: Methodology, Investigation, Data curation. Huixian Wang: Validation, Resources. Ruimin Wang: Writing – review & editing, Visualization, Supervision.

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.

Acknowledgments

This research was supported by First-class Discipline Breakthrough Initiative of Hainan University (No. XKTP2025B02) and Scientific Research Foundation of Hainan University (No. KYQD(ZR)23016).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2026.104254.

Appendix A. Supplementary data

Supplementary material 1

mmc1.docx (56KB, docx)

Supplementary material 2

mmc2.docx (1.1MB, docx)

Data availability

No data was used for the research described in the article.

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Associated Data

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Supplementary Materials

Supplementary material 1

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Supplementary material 2

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Data Availability Statement

No data was used for the research described in the article.

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