Anaerobic self-induced Fermentation: A Green Bioprocessing Strategy for Enhancing Extraction Efficiency and Bioactivities of Flavonoids from Erigeron breviscapus

doi:10.21203/rs.3.rs-7710172/v1

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Anaerobic self-induced Fermentation: A Green Bioprocessing Strategy for Enhancing Extraction Efficiency and Bioactivities of Flavonoids from Erigeron breviscapus

https://doi.org/10.21203/rs.3.rs-7710172/v1

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Erigeron breviscapus possesses anti-inflammatory and antioxidant properties, yet its therapeutic application is limited by the low yield of active components. Fermentation technology offers a promising solution to enhance bioactive enrichment. In this study, anaerobic self-induced fermentation was identified as the most effective approach among 12 distinct fermentation strategies evaluated. This method significantly increased the scutellarein yield by 20.35-fold under optimized conditions and reduced microbial α-diversity while inducing substantial community restructuring, characterized by a shift from Proteobacteria to Firmicutes in bacteria and from Ascomycota to Basidiomycota in fungi. Metabolomic analysis identified 223 differentially expressed metabolites, enriched primarily in flavonoid biosynthesis and phenylalanine metabolism pathways. Key bacterial genera such as Clostridium and Bacillus were strongly correlated with enhanced flavonoid accumulation. Furthermore, the anaerobically self-fermented extract exhibited significantly improved antibacterial and antioxidant activities. These findings demonstrate the efficacy of anaerobic self-induced fermentation as a targeted bioprocessing strategy to amplify the bioactivity and therapeutic potential of E. breviscapus.

Spontaneous Fermentation1

Erigeron breviscapus2

Microorganisms3

Flavonoids4

Antibacterial5

Antioxidant6

LC-MS7

Erigeron breviscapus (Vant.) Hand.-Mazz. (E. breviscapus) is a perennial herb native to southwestern China, renowned for its high scutellarin content—a flavonoid constituting over 85% of its total flavonoids(1). Studies have shown that Erigeron breviscapus flavonoids (EBF) exhibit remarkable effects in preventing cerebral ischemia-reperfusion injury, regulating angiogenesis, alleviating acute lung injury, and providing anti-inflammatory and antioxidant benefits(2). However, Conventional extraction methods for scutellarin and scutellarein remain inefficient, yielding limited quantities of these active compounds(3). Although chemical synthesis can achieve large-scale production, it is hindered by high raw material costs and stringent reaction conditions, making industrialization difficult. To overcome these limitations and meet the demands for both yield and safety, there is a pressing need to develop green and efficient bioprocessing technologies. Fermentation technology and enzymatic catalysis represent promising approaches to enhance the content and bioavailability of active ingredients in E. breviscapus.

In recent years, fermentation technology has been widely applied in the secondary development of traditional Chinese medicinal materials. By leveraging microbial activity, it enhances the content and bioavailability of active ingredients in medicinal materials, thereby boosting their biological activity(4). Fermentation enhances bioactive compound yield and efficacy. For instance, fermented Eucommia ulmoides leaves increased water-extract activity and alleviated hyperlipidemia in rats via gut-metabolism modulation (Duan & Guo, 2024). Similarly, Rhizopus oryzae fermentation of soybeans significantly raised phenols, flavonoids, and aglycone isoflavones while improving antioxidant capacity and gut microbiota regulation (Chen & Chen, 2023). Biotechniques like metabolic engineering enable targeted glycosylation to modify flavonoid properties (Xiao et al., 2014). Advanced tools including HPLC, LC–MS, 16S rRNA, and ITS sequencing now allow precise monitoring of microbial–substrate interactions during fermentation .

The present study aims to address the limitations in current extraction and synthesis methodologies for EBF by employing fermentation as a green bioprocessing strategy. We seek to optimize fermentation conditions to enhance flavonoid yield, characterize associated shifts in microbial community structure and metabolic profiles, and evaluate the functional improvements in antibacterial and antioxidant activities following fermentation. This work aims to contribute to the sustainable development and application of E. breviscapus as a high-value natural medicine.

Experimental Design

The experimental design was structured into three consecutive phases (Fig. 1). First of all, the screening and optimization of the most suitable fermentation process involved selecting microbial strains, single-factor condition optimization, and response surface methodology to establish a method that significantly enhanced scutellarein content. Secondly, changes in bacterial and fungal communities between the fermented (FJ group) and non-fermented (YY group) herb residues were analyzed using 16S rRNA and ITS sequencing, while LC-MS was employed to compare compositional profiles and investigate the mechanisms underlying the fermentation process. Finally, the influence of fermentation on the in vitro antibacterial efficacy of EBF against Escherichia coli, Shigella dysenteriae, Salmonella spp, and Staphylococcus aureus, as well as on antioxidant activity, was systematically evaluated.

Screening of Suitable Fermentation Methods

The experiment comprised 14 groups: seven probiotic-based groups (Lactobacillus plantarum, Bacillus coagulans, Clostridium butyricum, Bacillus subtilis, Penicillium cicariae, Bacillus licheniformis, and Aspergillus niger), three groups inoculated with local Yunnan yeast starters, and two self-induced fermentation groups (aerobic and anaerobic). One sterile control group and one untreated raw herb group were also included.

For probiotic and yeast starter groups, 10 g of E. breviscapus was sterilized (121°C, 30 min), cooled, mixed with 30 mL sterile water, and inoculated with 10 mL activated microbial culture, followed by fermentation at 37°C with shaking at 120 r/min. Self-induced fermentation groups used unsterilized herb mixed with 40 mL sterile water: aerobic fermentation was performed at 37°C with shaking (120 r/min), while anaerobic fermentation was conducted under paraffin seal at 37°C statically. The sterile control contained sterilized herb with 40 mL sterile water, shaken under the same conditions. The raw herb group remained unfermented.

After 5 days, all samples were freeze-dried, and total flavonoids were ultrasonically extracted. Groups showing significantly increased scutellarin and scutellarein levels by HPLC were selected for further optimization.

Screening and Optimization of Fermentation Conditions

A single-factor design was used to screen key parameters: solid-to-liquid ratio, fermentation time, temperature, nitrogen source, and carbon source addition. Optimal ranges were determined (see Table 1). Response surface methodology (RSM) via Design-Expert 8.0 software was then applied, with the above factors as independent variables (three levels each) and scutellarein content as the response. Coded factor levels are shown in Table 2. The product under optimal conditions was designated the FJ group, and the non-fermented raw material as the YY group.

Table 1
Experimental design for single-factor optimization of *Erigeron breviscapus* fermentation
Factor	Levels Tested	Fixed Conditions
		Tem	FT	SLR	Water
Solid-liquid ratio (SLR)	1:2; 1:4; 1:6; 1:8; 1:10; 1:12; 1:14	37°C	6d	/	Sterile
Fermentation time	2; 4; 6; 8; 10; 12; 14 d	37°C	/	1:8	Sterile
Temperature (°C)	28; 31; 34; 37; 40; 43; 46°C	/	6d	1:8	Sterile
Nitrogen source (Peptone)	0.4%; 0.6%; 0.8%; 1%; 2%; 3%; 4%	37°C	6d	1:8	Sterile
Carbon source (Glucose)	0.4%; 0.6%; 0.8%; 1%; 2%; 3%; 4%	37°C	6d	1:8	Sterile

Microbial Community Analysis via 16S rRNA and ITS Sequencing

Microbial DNA was extracted from membrane-filtered (0.2 µm) pre- and post-fermentation samples using an OMEGA Soil DNA Kit, followed by amplification of the 16S rRNA V3–V4 region (primers 338F/806R) and ITS1 region (primers ITS1F/ITS2R). Purified amplicons were sequenced on Illumina NovaSeq (PE250). Data were processed in QIIME2 for ASV generation and taxonomic assignment.

LC-MS-Based Metabolomic Profiling

Lyophilized samples were extracted with cold methanol/water (3:1) and analyzed using an EXIONLC System coupled with a QTrap 6500 + mass spectrometer. Chromatographic separation employed 0.1% formic acid in water (A) and acetonitrile (B). QC samples were pooled from each extract.

In Vitro Antibacterial and Antioxidant Assays

Antibacterial activity was tested against E. coli, S. dysenteriae, Salmonella spp., and S. aureus using well diffusion assays on LB agar with enrofloxacin as positive control. Antioxidant capacity was evaluated via T-AOC, DPPH, and ABTS assays (Boxbio, Beijing).

Statistical Analysis

Data are presented as mean ± SD from triplicate experiments. Statistical significance was assessed with one-way ANOVA (P < 0.05) in GraphPad Prism. MS data were processed using SCIEX Analyst Workstation and custom R scripts.

Establishment of the Fermentation Process for E. breviscapus

Through the screening of fermentation strains and methods, anaerobic self-induced fermentation was identified as the optimal approach, resulting in a 13.04-fold increase in scutellarein yield (Fig. 2A–C). Subsequent single-factor experiments determined the following optimal conditions: a liquid-to-material ratio of 1:8 (Fig. 2D), fermentation time of 6 days (Fig. 2E), temperature of 37°C (Fig. 2F), and supplementation with both nitrogen and carbon sources at 0.2% (Fig. 2G, H). Based on the Box-Behnken design (Fig. 3), the predicted optimal conditions were a liquid-to-material ratio of 1:8.39, fermentation time of 6.07 days, temperature of 36.59°C, nitrogen source at 0.19%, and carbon source at 0.22%, with a predicted scutellarein content of 28.10 mg/g. Validation under adjusted conditions (liquid-to-material ratio of 1:8.4, 6 days, 36.5°C, 0.19% nitrogen, 0.22% carbon) yielded a scutellarein content of 27.88 ± 0.60 mg/g, demonstrating a minimal deviation rate of 0.79% from the predicted value.

Anaerobic self-induced fermentation altered microbial abundance and community structure

The 16S rRNA sequencing method was employed to compare the microbial communities of spontaneously fermented and non-fermented (YY group) E. breviscapus herb residues. It was found that the spontaneous fermentation process significantly reduced the bacterial α-diversity (P < 0.05) (Fig. 4A). In terms of microbial composition, the YY group was dominated by Proteobacteria, with dominant genera including Bacteroides (60.73%-64.07%), Methylobacterium, and Pseudomonas. In contrast, the FJ group was dominated by Firmicutes (75.9%-76.7%), with Lactobacillus as the main dominant genus (75.44%-76.19%), and Bacteroides significantly decreased to 1.16%-1.28% (Fig. 4B, C). There were 2273 unique data points in the YY group, 566 in the FJ group, and 89 overlapping data points, indicating significant differences between the two groups (Fig. 4D). Heatmap analysis (Fig. 4E) revealed that in the YY group, Enterococcus, Brevibacterium, Bacillus, and Pseudobacillus had higher abundances, while in the FJ group, Escherichia-Shigella, Enterobacter, and Vibrio were the dominant genera. Functional prediction showed that microorganisms were active in amino acid synthesis, carbohydrate and lipid metabolism, and vitamin synthesis pathways. They possessed carbohydrate and lipid degradation capabilities, could utilize various carbon sources, and were involved in the degradation of aromatic compounds. Additionally, glycolysis and fermentation pathways were active, and they exhibited antibiotic resistance, electron transfer functions, and capabilities for nucleic acid metabolism and glycosylation (Fig. 4F).

Anaerobic self-induced fermentation altered fungal abundance and community structure

ITS analysis of fungal community changes before and after fermentation revealed that, similar to the bacterial community, fermentation significantly reduced fungal α-diversity (P < 0.05) (Fig. 5A). This was manifested in significantly lower Chao1 index, Observed_species, and Shannon index in the FJ group. In contrast, the Simpson and Pielou_e indices showed no significant differences, suggesting comparable species evenness between the two groups. The YY group was dominated by Ascomycota, followed by Basidiomycota, with the predominant genera being Knufia and Plectosphaerella (Figs. 5B). In contrast, the FJ group showed a significant increase in Basidiomycota and a notable decrease in Ascomycota, along with an increase in Zygomycota (Figs. 5C). The dominant genera in the FJ group were Rhodotorula and Coprinopsis. The Venn diagram underscores a striking dominance of unique fungal species in the FJ group, which boasts 77 unique data points compared to the YY group's 218. With a mere 9 shared points, this stark contrast highlights an extremely significant divergence in fungal community structures between the two groups. (Fig. 5D). The FJ group was uniquely defined by a high abundance of Rhodotorula and Coprinopsis, contrasting with the Knufia-dominated YY group (Fig. 5E). Additionally, the abundance of Fusarium and Phallus increased in the FJ group. Functional predictions indicate that fermentation significantly enhanced microbial metabolic functions, including amino acid and fatty acid synthesis, carbohydrate degradation, and energy metabolism (Fig. 5F). Fermentation improved energy generation pathways such as electron transport, glycolysis, and the tricarboxylic acid (TCA) cycle. Furthermore, nucleotide metabolism was enhanced, increasing microbial metabolic flexibility and energy production capacity.

Anaerobic Self-Induced Fermentation Alters Metabolites and Metabolic Pathways

Principal component analysis (PCA) showed that samples from FJ and YY were significantly separated along the PC1 dimension, with QC samples concentrated at the center of the plot, indicating good experimental stability and data reliability (Fig. 6A). Metabolomics analysis identified 223 differential metabolites, including 156 upregulated (such as gibberellin A7, wogonin, apigenin, hesperidin, daidzein, and tetrahydropalmatine) and 67 downregulated (such as abscisic acid, narcissus glycoside, jasmonic acid, rutin, L-valine, emodin, and adenosine). These differential metabolites span 50 categories, including plant hormones, flavonoids, amino acids and their derivatives, organic acids, alkaloids, and sphingolipids (Fig. 6B). KEGG pathway enrichment analysis revealed that the main metabolic pathways include cofactor biosynthesis (11.01%), ABC transporters (11.01%), flavonoid biosynthesis (8.26%), phenylalanine metabolism (6.42%), tyrosine metabolism (5.5%), flavonoid and isoflavonoid biosynthesis (5.5%), and arginine biosynthesis (3.83%) (Fig. 6C).

Correlation analysis between microbial communities and compounds

To clarify the impact of microorganisms on metabolism before and after fermentation, we performed a correlation analysis between the two, and the results showed that Clostridium was significantly positively correlated with wogonin, possibly enhancing flavonoid production through phenylalanine and flavonoid metabolic pathways; Bacillus was positively correlated with baicalin, possibly enhancing accumulation by promoting substrate conversion (Fig. 6D). Network analysis indicated that Clostridium and Bacillus are key collaborative microorganisms that jointly promote the metabolism of flavonoid precursors and two of them promote the metabolism of flavonoid precursors through synergistic action (Fig. 6E).

Fermentation of E. breviscapus Extract Significantly Enhances In Vitro Antibacterial and Antioxidant Activities

To investigate the effect of fermentation on the bioactivity of E. breviscapus flavonoid extracts, we conducted in vitro antioxidant activity assays and antimicrobial activity evaluations using extracts of the fermented products. The results indicate that fermentation significantly enhanced the antimicrobial activity of the original extract against Escherichia coli, Shigella dysenteriae, Salmonella, and Staphylococcus aureus. In FJ group, the inhibition zone diameters were 23.04 ± 0.95 mm for Escherichia coli, 23.82 ± 1.15 mm for Shigella dysenteriae, 20.95 ± 0.47 mm for Salmonella, and 21.35 ± 0.21 mm for Staphylococcus aureus, all significantly higher than the original extract group, and close to or exceeding the effect of the positive control group. In contrast, after high-pressure treatment, the inhibition zone diameters of the four bacteria were generally reduced, suggesting that high-pressure treatment may weaken the antimicrobial activity (Fig. 7A-E). In the in vitro antioxidant experiments, both the YY group and the FJ group showed significantly enhanced antioxidant capacity with increasing concentration. The antioxidant capacity of the FJ group was significantly superior to that of the YY group in tests for A-TAC, ABTS radical scavenging rate, and DPPH radical scavenging rate (Fig. 7F-H). The results suggest that fermentation treatment significantly improved the antioxidant capacity of the samples, demonstrating stronger free radical scavenging effects and higher total antioxidant potential.

Table 2 Fermentation process response surface test factor level table

Variable	Coded	Levels
Variable	Coded	-1	0	1
Solid-liquid ratio (SLR)	A	6	8	10
Fermentation time (d) (FT)	B	5	6	7
Temperature (°C)	C	34	37	40
Nitrogen source (Peptone) (%)	D	0	0.2	0.4
Carbon source (Glucose) (‰)	E	0	0.2	0.4

Fermentation is an important technique in the processing of traditional Chinese medicines. Under appropriate conditions of temperature, humidity, and moisture, the fermentation of medicinal herbs by microorganisms enhances the original properties of the herbs or produces new pharmacological effects, expanding the uses of traditional Chinese medicines to meet the strict requirements of clinical applications^[7]. In this experiment, optimization of the extraction process revealed that anaerobic spontaneous fermentation significantly increased the content of active ingredients in E. breviscapus. Similar to the anaerobic fermentation research on coffee (5), fermentation notably enhanced the content of flavonoid compounds, and the results were consistent with subsequent LC-MS detection. The experimental results showed that, except for Saccharomyces cerevisiae C, the fermentation of E. breviscapus with nine selected exogenous microorganisms led to varying degrees of reduction in scutellarin and its aglycone. Although five new absorption peaks were observed in the chromatogram, these peaks also appeared in the sterile control group, suggesting that the high-temperature, high-pressure sterilization process led to the decomposition and loss of components. After spontaneous anaerobic fermentation, the scutellarin content in the original medicinal material significantly decreased. Structurally, scutellarin aglycone is the aglycone portion of scutellarin with the glucuronic acid group removed, indicating that scutellarin likely underwent hydrolysis during the spontaneous fermentation process, with the revival of the E. breviscapus 's endogenous microbiota and various enzymes. The self-induced anaerobic fermentation method used in this experiment is simple to operate, cost-effective, and environmentally friendly, making it a promising approach for further development and utilization.

Through the analysis of the microbial community structure before and after anaerobic spontaneous fermentation of E. breviscapus, it was found that fermentation significantly altered the bacterial and fungal communities in the herb. In the bacterial community, Firmicutes dominated the fermentation group, similar to the findings in Jyoti Prakash Tamang's study of Korean fermented soybeans(6), where the abundance of Enterococcus, Escherichia-Shigella, and Paenibacillus increased. Enterococcus and Paenibacillus are considered highly potential microorganisms in food fermentation, especially in fermented vegetables, dairy products, and fermented meats(7). These microorganisms can modify compounds through metabolic enzymes such as deglycosylation, methylation, and hydroxylation. Therefore, the increase in scutellarin aglycone may be due to the deglycosylation of scutellarin, transforming the glycoside into the aglycone without the sugar moiety. In the original medicinal material group, Proteobacteria dominated, with Pediococcus being the primary genus. Pediococcus, a type of white rot fungus, is widely distributed in nature, particularly in decaying wood environments. As an important wood decomposer, Pediococcus strains secrete various degrading enzymes, especially ligninolytic enzymes such as laccase, lignin peroxidase, and manganese peroxidase, playing a significant role in breaking down lignocellulosic compounds (lignin, cellulose, and hemicellulose). It is hypothesized that the presence of Pediococcus is mainly due to the use of dried whole E. breviscapus before fermentation, which led to self-proliferation during the storage process.The fungal community also showed significant changes. In the original medicinal material group, Ascomycota dominated, with Xenodidymella and Plectosphaerella as the main genera, typically saprophytic fungi found in plant residues involved in the decomposition of organic matter. The fermentation group, on the other hand, was dominated by Basidiomycota, with Rhodosporidiobolus, Coprinellus, and Fusarium as the primary genera. Research by Silvia Donzella(8) found that Rhodosporidiobolus azoricus utilizes low-value industrial food waste (such as pumpkin skins and syrup from preserved fruit processing) to produce yeast biomass and lipids, which aligns with the oily texture of the fermentation product and the abundance of Rhodosporidiobolus azoricus at the phylum level in our observations. Yu Yang's research(9) found that Coprinellus disseminatus effectively degrades plant tissues by secreting various enzymes such as xylanase (XLE), cellulase (CLE), acetyl xylan esterase (AXE), and α-L-arabinofuranosidase (α-L-AF). Fusarium, a genus rich in secondary metabolites, has unique chemical diversity. The analysis of its biosynthetic gene clusters (BGCs) shows that this genus has abundant untapped genetic potential, capable of producing a wide variety of novel secondary metabolites(10).Overall, fermentation profoundly affects the microbial community structure, likely due to changes in oxygen conditions, temperature, and nutrient availability during fermentation. Lactic acid bacteria (Enterococcus—a genus in the Lactobacillales order) possess the ability to biotransform flavonoid compounds. They can secrete various enzymes to remove the glycosidic part of flavonoids, generating flavonoid aglycones with higher biological activity.

Through the analysis of the components of E. breviscapus before and after anaerobic spontaneous fermentation, the metabolic profiling comparison between Group A (fermented) and Group B (non-fermented) revealed significant changes. Upregulated metabolites in the fermented group include Gibberellin A7, Genistein, Diosmetin, Glycitin, L-Histidine, and Fumaric acid, while downregulated metabolites include (+)-Abscisic acid, Narcissoside, (±)-Jasmonic acid, Rutin, L-Valine, and Emodin.Bruno Dias Nani(11)'s study found that Gibberellin A7 (GA7) exhibits significant antifungal, antibiofilm, and antioxidant activities with low toxicity, showing promising safety and potential applications in infection control. Clinical studies have also reported various biological effects of Genistein, such as antioxidant, anti-inflammatory, antibacterial, antiviral activities, angiogenesis, estrogenic effects, and pharmacological activity on diabetes and lipid metabolism(12). Lihua Zhao(13)'s research found that Diosmetin can activate the SIRT1 pathway to exert anti-inflammatory and ferroptosis-reducing effects, alleviating pathological changes caused by Staphylococcus aureus-induced mastitis.Saghi Hakimi Naeini(14)'s research shows that Glycitin modulates oxidative stress markers and activates the Nrf2/HO-1 signaling pathway, exhibiting anticonvulsant and neuroprotective effects. Mengze Li(15)'s study found that L-Histidine inhibits Gab2 expression, reduces nitric oxide (NO) production, lowers inflammatory cytokines such as IL-6 and IL-8, alleviates non-esterified fatty acid (NEFA)-induced inflammation, and restores cell proliferation, showcasing its potential therapeutic mechanism for NEFA-related metabolic disorders. Fumaric acid, a critical organic acid in the tricarboxylic acid (TCA) cycle, plays a vital role in cellular energy metabolism and is used in pharmaceutical fields to treat diseases such as psoriasis, with anti-inflammatory and immune-modulatory effects(16).Ru-Huei Fu(17)'s research found that Narcissoside can activate the miR200a/Nrf2/GSH antioxidant pathway, reducing oxidative stress and cell apoptosis, and protecting dopaminergic neurons, showing neuroprotective effects against Parkinson's disease. Rutin, a flavonoid widely present in many plants, has various biological activities, including anti-inflammatory, antioxidant, neuroprotective, nephroprotective, and hepatoprotective effects(18).After fermentation, the content of flavonoid compounds significantly increased, with antioxidants and antimicrobial components like Gibberellin A7, Genistein, and Diosmetin significantly upregulated. The correlation analysis between the microbiota and these compounds revealed that Clostridium was positively correlated with flavonoids, likely enhancing flavonoid production via phenylalanine and flavonoid metabolic pathways. Bacillus was positively correlated with Glycitin, possibly enhancing accumulation by promoting substrate conversion, while Escherichia was negatively correlated with flavonoids, potentially affecting flavonoid production through competition for substrates or metabolic inhibition. Network analysis indicated that Clostridium and Bacillus are key collaborating microorganisms that jointly promote the metabolism of flavonoid precursors, while Escherichia inhibits flavonoid production through competitive inhibition.

Fermentation not only altered the chemical composition and microbial community structure but also affected the antimicrobial and antioxidant activities of E. breviscapus. Currently, research on the antimicrobial effects of flavonoids from E. breviscapus is still limited. In this study, by comparing the antimicrobial activity of flavonoids extracted from E. breviscapus before and after fermentation, it was found that fermentation significantly enhanced the antimicrobial activity. The antibacterial effect of the fermented flavonoid extract was on the same level as that of the positive control, ofloxacin, based on the inhibition zone diameter. This indicates that the fermented flavonoid extract from E. breviscapus shows great potential for antimicrobial activity, warranting further exploration of its antibacterial effects and mechanisms. Huijing Zhou(19)'s study found that fermentation of Salvia miltiorrhiza roots with Aspergillus flavus significantly improved their antioxidant and antimicrobial activities, converted into more polar compounds, and enhanced bioactivity, which is similar to the findings in this experiment. During fermentation, bacteria like lactic acid bacteria can synthesize vitamins and minerals, generate bioactive peptides through enzymes such as proteases and peptidases, remove some antinutritional factors, and exhibit various effects, including antioxidant, antimicrobial, anti-allergic, and blood pressure-lowering activities(20). Although this experiment only provided a preliminary exploration of the antimicrobial and antioxidant activities of fermented E. breviscapus products, the results indicate a significant potential for these properties, suggesting that further research is warranted.

This experiment significantly increased the content of active ingredients in E. breviscapus, especially the content of E. breviscapus glycosides, by optimizing the conditions for anaerobic spontaneous fermentation. Although the data obtained by HPLC is already convincing, there is a small regret in the subsequent metabolomics analysis, as the database of the testing institution does not include E. breviscapus and its glycosides. During fermentation, the changes in the microbial community had a profound impact on the chemical composition and biological activity of E. breviscapus. Specifically, the fermentation promoted the abundance of microorganisms such as lactic acid bacteria, Enterococcus, and Bacillus, which may have catalyzed the biotransformation of flavonoids through metabolic enzymes. Additionally, metabolomics analysis indicated that fermentation significantly regulated the synthesis of various metabolites, especially flavonoids. This experiment also preliminarily explored the antioxidant and anti-inflammatory activities of fermented E. breviscapus, and the results showed that fermentation significantly enhanced its biological activity. However, further research is needed to verify these biological effects and their mechanisms. Although some component loss and changes in microbial community structure were observed due to high-temperature and high-pressure sterilization, the self-induced anaerobic fermentation method still showed advantages such as simplicity, low cost, and environmental friendliness. Future research could further optimize fermentation conditions and explore the synergistic mechanisms of different microorganisms to develop more efficient traditional Chinese medicine fermentation technologies and expand the clinical applications of E. breviscapus and other medicinal herbs.

This study demonstrated that fermentation significantly enhances the bioactivity and chemical composition of E. breviscapus. Spontaneous fermentation optimized through response surface methodology increased scutellarein aglycone content by 20.35-fold and reshaped microbial communities, enriching beneficial taxa like lactic acid bacteria and Rhodotorula aurantiaca. LC-MS analysis identified 223 differential metabolites, with key pathways such as flavonoid biosynthesis significantly enriched. Fermentation improved the antibacterial and antioxidant activities of E. breviscapus, showcasing its potential as a functional natural product. These findings provide a scientific basis for further applications of fermentation technology in enhancing the value of traditional Chinese medicine.

Conflict of Interest

The 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

This article is supported by the Yunnan Province Major Agricultural Science and Technology Project: "Innovation and Demonstration of Key Technologies for Healthy Pig Farming and Waste Recycling in Weixin County" (202202AE090027);Yunnan Province Rural Revitalization Science and Technology Project: "Science and Technology Special Taskforce for Circular Agriculture in Weixin County, Yunnan Province" (202304BI090011);Yunnan Provincial Key Laboratory of Animal Epidemic Pathogen Biology (202449CE340019).

Author Contribution

Qianfei Wei, Xue Zhang, Qiong Pan: Original draft, Methodology, Visualization, Investigation and Data curation. Ying Zhang , Shanqiang Wang , Dayong Wang: Methodology, Visualization. Chunlian Song , Xianghua Shu : Writing-original draft, Writing-Review. Chunlian Song and Xue Zhang: Writing-Review. Xianghua Shu: Project administration.

Acknowledgments

The paper was coauthored by Qianfei Wei, Chunlian Song

Data Availability Statement

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (21) in National Genomics Data Center(22), China National Center for Bioinformation at Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA023872) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/s/h6K6lUAa

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Anaerobic self-induced Fermentation: A Green Bioprocessing Strategy for Enhancing Extraction Efficiency and Bioactivities of Flavonoids from Erigeron breviscapus

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1 Introduction

2 Materials and Methods

3 Results

4 Discussion

5 Conclusion

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