Synergistic Enhancement of Antioxidant and Cytoprotective Activities in Lonicerae Japonicae Flos via Co-fermentation: Metabolomics Unraveling Key Biotransformation Pathways

Synergistic Enhancement of Antioxidant and Cytoprotective Activities in Lonicerae Japonicae Flos via Co-fermentation: Metabolomics Unraveling Key Biotransformation Pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic Enhancement of Antioxidant and Cytoprotective Activities in Lonicerae Japonicae Flos via Co-fermentation: Metabolomics Unraveling Key Biotransformation Pathways Yiwen Wang, Dengfan Lin, Shaowei Yan, Chang Gao, Zuohua Zhu, Wenbing Gong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7563014/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract ABSTRAT Lonicera japonica is a medicinal food rich in polyphenols. However, most of the polyphenols are in the bound state, thus resulting in low extraction efficiency. In this study, the antioxidant and cytoprotective activities of L. japonica were improved after fermentation, through metabolomics, it was found that the level of some polyphenols increased, which revealed the reason for the enhanced antioxidant and cytoprotective activities of L. japonicum after fermentation. Results showed that the ·OH scavenging rate of Picp-2 and co-fermentation groups increased of 23% and 13%, the DPPH· scavenging rate of SY group increased of 10%, and the cytoprotection activity of co-fermentation group increased of 32%. Besides, metabolomics based on LC/MS identified a total of 576, 358, 651 differential metabolites in Picp-2, SY and co-fermentation groups respectively. The levels of benzoic acids, coumaric acids, cinnamic acids, fatty acids and flavonoids in Picp-2 and SY groups significantly increased. The Picp-2 group separately detected the increase of amino acids, quinic acids, terpene glycosides, and catechin. The changes of metabolites level in co-fermentation group were similar to the single strain groups, while the change fold was smaller. Moreover, the correlation analysis revealed a significantly positive correlation between the contend of demethyltexasin, ferulic acid, chlorogenic acid, and eriodictyol with antioxidant activity. Additionally, the amount of taxifolin, paeonol, and riboflavin were significantly positively correlated with cytoprotection activity. This study is the first to find that co-fermentation with Lactiplantibacillus plantarum and Saccharomyces cerevisiae could improve the cytoprotective activity of L. lonicerae , and revealed the potential biotransformation pathway of active substances through metabolomics. Biotransformation Lactiplantibacillus plantarum Lonicerae japonicae flos Metabolomics Saccharomyces cerevisiae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Lonicerae japonicae flos was the dried bud or flower of Lonicera japonica Thunb. It was a traditional medicine material that belongs to the category of medicine food homology [ 1 ]. Modern pharmacological researches had demonstrated that the extracts from L. japonicae flos possessed a diverse range of biological activities, such as antioxidant, antimicrobial, antitumor, antiviral, and immunomodulatory effects [ 2 – 4 ]. The L. japonicae flos was also used to prevent COVID-19, on account of the inhibition of SARS-CoV-2 M pro activity, thereby alleviating viral entry as well as replication [ 5 ]. In previous reports, over 200 active compounds had been isolated from L. japonicae flos, including phenols, organic acids, flavonoids, monoterpenoids, triterpenoids, volatile oils and lignans [ 1 – 3 ]. However, these active compounds were typically bound to cellular structures, such as hemicellulose, xylan, cellulose and lignin. Therefore, the extraction methods of active compounds in L. japonicae flos, including water extraction, ultrasonic alcohol extraction and enzyme-assisted extraction, had a limited extraction efficiency [ 6 – 8 ]. Fermentation was a common biotransformation method, which could liberate active compounds bound to the cellular structures and improved their extraction efficiency [ 9 ]. It was reported that after the co-fermentation of lactobacillus and yeast, the phenols and flavonoids of brown rice increased by 93.3% and 61.3%, respectively [ 10 ]. Furthermore, fermentation could leverage the metabolic activities of microorganisms to convert active compositions in the substrate, augment its original efficacy and generate novel effects. For example, the value of FRAP and DPPH· scavenging rate of fruit juice fermented by Lactobacillus plantarum , Lactobacillus salivarius , and Saccharomyces boulardii were significantly increased compared with the control group [ 11 ]. Therefore, fermentation could enhance the scope of application and utilization of plant materials, and develop more valuable products. However, only a few studies had been focused on the fermentation of L. japonicae flos, let alone on the metabolites changes during the fermentation. Untargeted metabolomics based on LC-MS provided a comprehensive overview of all known and unknown metabolites, thereby justifying the potential active compounds biotransformation during fermentation [ 12 , 13 ]. Li et al. utilized the untargeted metabolomics to isolate 66 volatile compounds and 30 nonvolatile compounds of L. japonicae flos processed with different drying methods [ 14 ]. In another study, metabolomics had found 27, 81, 113 differential metabolites in flowers, flower buds, leaves of L. japonicae Thunb [ 15 ]. Overall, untargeted metabolomics based on LC-MS proved to be an effective approach for elucidating the metabolic changes during L. japonicae flos fermentation. Above all, this study aim to improve the extraction efficiency of active substances in L. japonicae flos by using a novel biopretreatment method as well as improve the antioxidant and cytoprotection activities of L. japonicae flos after fermentation, and metabolomics is also used to reveal the reasons for the changes in substances and activities at the molecular level. This study could provide a reference for the biotransformation of metabolites in L. japonicae flos, facilitating the development of new products with improved functionality fermented L. japonicae flos. 2. Materials and methods 2.1. Chemicals and standards 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2´-azino-bis (3-ethylb-enzothiazoline-6-sul-fonic acid) (ABTS), Folin-Ciocalteu reagent, gallic acid were purchased from Sigma Chemicals Co. (St Louis, MO, USA). 3,5-dinitrosalicylic acid, ascorbic acid, gallic acid, rutin, coomassie brilliant blue, vitamin E, D-glucose were purchased fromo Merk (Darmstadt, Germany). Celluclast® (700 U/g) was purchased from Novozymes (China) Biotechnology Co., Ltd (Tianjin, China). 2.2. Samples and microorganism collection Lonicerae japonicae flos was purchased from Hunan Longhui Yi-du Se-enriched Tea Industry Co., Ltd. (Hunan, China). Lactiplantibacillus plantarum Picp-2 (CCTCC M20191045) was purchased from the China Center for Type Culture Collection (Wuhan, China). Saccharomyces cerevisiae SY (CGMCC 2.119) was purchased from China General Microbial Culture Preservation and Management Center (Beijing, China). 2.3. Sample processing Ten grams of L. japonicae flos powder (crushed and filtered through a 60-mesh sieve) were mixed with 200 mL of 630 U/mL cellulase solution (0.45%, w/v). Then the mixture was put into a constant temperature oscillation box at 50℃ and stirred for 6 h at 180 rpm. The mixed enzymatic hydrolysate of the L. japonicae flos was prepared for further fermentation. 2.4. Preparation of fermented Lonicerae japonicae flos The enzymatic hydrolysate of the L. japonicae flos (as described in 2.3) was sterilized at 121℃ for 15 min. The cells were then inoculated into 250 mL conical flasks containing 200 mL enzymatic hydrolysate of L. japonicae flos with a population of 2% S. cerevisiae SY and 10% L. plantarum Picp-2, in both single and co-culture groups, then incubated at 37℃ for 72 h [ 11 ]. The initial microbial populations were established based on previous researches conducted on fermented beverages. After fermentation, they were centrifuged twice (7000 rpm, 4℃, 5 min) and the supernatant was gathered and stored at -20℃ for further analysis. 2.5. Determination of antioxidant activities In vitro antioxidant activities of fermented broth were evaluated in terms of the scavenging rate of 1,1-diphenyl-2-picryl-hydrazyl radicals (DPPH·), 2,2´-azino-bis (3-ethylb-enzothiazoline-6-sulfonic acid) radicals (ABTS ·+ ), hydroxyl radicals (·OH) and ferric reducing antioxidant power (FRAP) assay as described [ 16 ], the ascorbic acid (0.8 mg/mL) was used as a positive control. 2.6. Determination of active compositions content 2.6.1. Determination of total phenols content The total phenols content in the fermented broth was quantified using Folin-Ciocalteu reagents with gallic acid as the reference standard. [ 17 ]. One milliliter of fermented broth was diluted by a factor of 10 and mixed with 1.5 mL of Folin-Ciocalteu reagent for 3–8 minutes at room temperature. Then 1 mL of 20% (w/v) Na 2 CO 3 was added to the mixtures and adjusted final total volume to 10 mL with 6.5 mL of dH 2 O. The mixtures were incubated for 1 h to facilitate the chromogenic reaction. Afterwards, their optical density was determined at a wavelength of 760 nm. The total phenols content was calculated according to the gallic acid calibration curve ( R 2 = 0.9959) and expressed as mg gallic acid equivalents (GAE)/mL. 2.6.2. Determination of total flavonoids content The total content of flavonoids in the fermented broth was quantified using a colorimetric method with rutin as the reference standard [ 18 ]. One milliliter fermented broth was mixed with 12 mL of 75% (v/v) ethanol and 1 mL of 5% (w/w) NaNO 2 for a duration of 5 min, followed by the addition of 1 mL of 10% (w/w) Al(NO 3 ) 3 . After a 5-minute incubation period, 10 mL of NaOH solution (1 M) was introduced into the mixture. The solution was reacted for 15 min. Afterwards, the absorbance of the mixture was measured at 510 nm. The total flavonoid content was calculated from the calibration curve of rutin standard solution ( R 2 = 0.9912) and expressed as mg rutin equivalents (RTE)/mL. 2.6.3. Determination of reducing sugars content The quantification of reducing sugars were performed using the 3,5-dinitrosalicylic acid (DNS) method [ 19 ]. The DNS reagent was prepared by dissolving 1 g of DNS and 30 g of potassium sodium tartrate in 80 mL of 0.5 M NaOH at a temperature of 45℃ with vigorous stirring until complete dissolution. After cooling to room temperature, the solution was diluted with ddH 2 O to a final volume of 100 mL. For the measurement, 2 mL of DNS reagent were added to a tube containing 1 mL of fermented broth and incubated at 95℃ for 5 minutes. After cooling, the solution was diluted with 7 mL of distilled water and the absorbance was measured at 540 nm using a microplate reader. The reducing sugar content was determined using a calibration curve of standard glucose ( R 2 = 0.9975) and expressed as mg glucose equivalent (GE)/mL. 2.6.4. Determination of total proteins content The quantification of total proteins were performed using the Bradford method. Twenty µL of fermented broth were mixed with 200 µL of coomassie brilliant blue (CBB). After 10 min, the absorbance of the mixture was measured at 595 nm in a microplate reader. The protein concentrations were determined according to the absorbance rate of the sample and a 300 mg/L bovine albumin. 2.7. Cytoprotection activity of fermented Lonicerae japonicae flos To evaluate the protective efficacy of fermented L. japonicae flos against oxidative stress induced by H 2 O 2 . [ 20 ], human keratinocytes were pretreated with unfermented and fermented L. japonicae flos for 30 min. H 2 O 2 was then added (0.5 mmol/L in HaCaT) for 4 h. For each experiment, the controls were prepared using the same concentration of DMSO as the samples and containing an appropriate volume of H 2 O 2 /sterile water along with vitamin E. (300 µg/mL) was used as a positive control. After treatment, the protective effect of fermented L. japonicae flos was estimated by neutral red uptake assay. Neutral red (NR) at a concentration of 0.03% (m/v) in phosphate-buffered saline (PBS) was introduced to the cells. The human keratinocytes were incubated for an additional 2 hours at 37℃, then washed with a formaldehyde (0.125%, v/v) and CaCl 2 (0.25%, m/v) mixture before dissolving NR retained in the cells using acetic acid (1%, v/v) in methanol (50%, v/v). The plates were read on a microplate reader at 540 nm. 2.8. Untargeted metabolomics analysis 2.8.1. Preparation of fermented Lonicerae japonicae flos metabolome samples The fermented L. japonicae flos was freeze dried in a lyophilizer. The sample was subsequently dissolved in a mixture of acetonitrile and methanol (8:2 v/v) at a solid-liquid ratio of 1:50 (w/v) [ 21 ]. Samples were then centrifuged at 8000 rpm for 5 min. The supernatants were filtered through 0.22 µm filter membrane. Mixed all extracts to be a pooled sample as a quality control (QC) sample. 2.8.2. Metabolomics analysis by LC-MS Conditions for ultra performance liquid chromatography (UHPLC) analysis involved the use of Agilent 1200 UHPLC system (Waldbronn, Germany) with a SunFireTM C18 chromatographic column (250 x 4.6 mm, 5 microns, Waters, USA). The metabolic compounds were detected by Q-Exactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) under two ion modes (positive-ion and negative-ion modes). The gradient elution procedure was conducted with eluent A (0.1% formic acid in water, v/v) and eluent B (Acetonitrile). The elution procedure was 0–5 min, 15% B; 5–10 min, 15–20% B; 10–20 min, 20–25% B; 20–30 min, 25–35% B; 30–40 min, 35–50% B; 40–50 min, 80% B; For 50–55 minutes, 15% B. The flow rate was maintained at 0.3 mL/min. The column temperature was kept at 30 ℃, and the sample injection volume was set at 5 µL. Q-Exactive quadrupole-Orbitrap mass spectrometer was operated at capillary temperature of 320℃, sheath gas flow rate of 35 psi and aux gas flow rate of 10 L/min, aux gas heater temperature of 350℃. The scan range covered 50-1000 m/z in the negative-ion mode and 50-2000 m/z in the positive-ion mode. 2.9. Metabolomics data processing The LC-MS/MS data were processed and analyzed using multivariate techniques, as previously described [ 22 ]. Principal component analysis (PCA) and t-test were used to identify the differential metabolites among the samples. Metabolites with variable importance in projection (VIP) > 1.0 and a P < 0.05 were considered to be differential metabolites. The heatmaps were created on OeBiotech ( https://cloud.oebiotech.com/task/ ). 2.10. Statistical analysis All measurements were performed in triplicate. Results in the present study were shown as means. Statistical comparisons were made using a two-way analysis of variance (ANOVA) with Tukey’s post hoc test. P < 0.05 were considered as statistical significance. Columns with different letters indicate a significant statistical disparity. Correlation analysis was performed by using a two-tailed Pearson's correlation test of GraphPad Prism version 9.4 software package. 3. Results and discussion 3.1. The pH and cell count in Lonicerae japonicae flos fermented broth The utilization of a combination of lactobacilli and yeast for fermentation had been widely reported [ 23 , 24 ], with much publication dedicated to their interactions [ 25 ], However, there are still no reports of adopting this fermentation strategy in Lonicerae japonicae flos. In present study, compared with the unfermented group, the pH of Picp-2 and Picp-2 + SY groups were decreased to 3.65–3.89 (Fig. 1 A). Moreover, the count of L. plantarum Picp-2 in Picp-2 + SY group (6.36 log CFU/mL) was significantly higher ( P < 0.05) than that in Picp-2 group (6.07 log CFU/mL). As was widely recognized, yeast could produce ethanol and CO 2 , which induced the stress response in lactobacilli, thereby enhancing thermal tolerance and promoting the biosynthesis of unsaturated fatty acids [ 26 , 27 ]. The CO 2 could also create an anaerobic environment that promote the growth of lactobacilli [ 28 ]. Meanwhile, yeast could metabolize and produce amino acids to promote the growth of lactobacilli [ 29 ]. As opposed to lactobacilli, the count of S. cerevisiae SY in Picp-2 + SY group was significantly lower ( P < 0.05) than that in SY group. Previous study showed that the lactobacilli could produce a mass of acid, which result in an extremely low pH environment, that effectively suppressed yeast growth [ 29 ]. This result indicated that S. cerevisiae could promote the growth of L. plantarum . Conversely, L. plantarum inhibited the growth of S. cerevisiae . Therefore, co-fermentation is beneficial to promote the metabolic activity of the single strain, so that it can more actively interact with the fermentation substrate and improve the extraction efficiency of active substances. 3.2. The contents of phenols, flavonoids, reducing sugars and proteins in Lonicerae japonicae flos fermented broth Compared with the unfermented group, the total phenols content in the Picp-2 and Picp-2 + SY groups reduced from 59.67 mg GAE/mL to 51.99 and 53.09 mg GAE/mL ( P < 0.05), respectively (Fig. 1 B). The changing of total flavonoids content in these groups was similar to that of total phenols content. In addition, the content of reducing sugars in Picp-2, SY and Picp-2 + SY groups was reduced to 0.23–0.29 mg GE/mL ( P < 0.05), and the total protein content in Picp-2, SY and Picp-2 + SY groups was decreased to 0.18–0.20 mg/mL ( P < 0.05, Fig. 1 C). There was some controversy regarding the release of substances from substrates by fermentation. Some reports showed that the microorganism could promote the release of phenols and flavonoids of substrate, while in another study, microorganism could consume and reduce the phenols and flavonoids during fermentation [ 30 ]. Our result showed that fermentation with Picp-2 and SY could reduce the content of phenols and flavonoids in L. japonicae flos. 3.3. Antioxidants and Cytoprotection activities in Lonicerae japonicae flos fermented broth Compared with the unfermented group (69.02%), the ·OH scavenging rate of the Picp-2 (85.06%) and Picp-2 + SY (77.88%) groups showed a significant increase ( P < 0.05 ), while the SY group (65.28%) had not significant change (Fig. 2 A). The DPPH· scavenging rate of the SY group was significantly increased to 85.43%, nevertheless, the Picp-2 and Picp-2 + SY groups did not exhibit significant differences (Fig. 2 A). The ABTS ·+ scavenging rate of fermtation groups had no significant differences after fermentation. The FRAP of the SY group (1.026) was significantly increased compared with the unfermented group (1.002). On the contrary, the FRAP of Picp-2 group (9.708) was significantly decreased after fermentation, and the Picp-2 + SY group had not significantly changed (Fig. 2 A). Bsides, there was also an increase in cytoprotection of L. japonicae flos, compared with the unfermented group, the cell viability of the Picp-2 + SY group treated human keratinocytes significantly increased from 49.13% to 64.94%. However, the cell viability of Picp-2 (51.33%) and SY (53.53%) groups treated human keratinocytes had no significant difference compared with unfermented group (Fig. 2 B). This result indicated that the fermentation with Picp-2 and SY could improve the antioxidant and cytoprotection activities of L. japonicae flos. The most use of L. japonicae flos is for drinking and has strict requirements for "large white flower" and "two white flower" buds, which limits its use. This study removed the limitation of flowering period and enhance the antioxidant and cytoprotective activities of L. japonicae flos, which provides a broader market prospect for its development and application. 3.4. Metabolic profile in Lonicerae japonicae flos fermented broth To gain a comprehensive understanding of the changes of metabolites during L. japonicae flos fermentation, the L. japonicae flos fermented broth and the unfermented group were chosen for the following metabonomics analysis. A total of 1082 and 442 metabolites were identified in positive and negative ion mode, respectively, mainly including phenols, flavonoids, amino acids, peptides, sugars, organic acids and saponins. PCA analysis showed that the unfermented and fermented L. japonicae flos could separate well from each group and differential metabolites analysis of the metabonomics could be performed (Fig. 3 ). 3.5. Differential metabolites analysis of Lonicerae japonicae flos fermented broth The volcano plot could clearly depict the significant situation of differential expression of metabolites. A total of 576, 358, 651 differential metabolites was identified in Picp-2, SY, Picp-2 + SY groups, respectively. Among them, 274, 119, 301 metabolites were up-regulated in Picp-2, SY, Picp-2 + SY groups, along with 302, 239, 350 metabolites were down-regulated (Fig. 4 A B C). Compared with unfermented groups, the expression levels of benzoic acids, coumaric acids, cinnamic acids, fatty acids and flavonoids in the Picp-2 and SY groups were significantly increased. The exclusive increase of steroidal saponins, amino acids, alkaloids, quinic acids, terpene glycosides and catechin was observed in the Picp-2 group. The level of triterpenoid saponins was only increased in SY group. 3.5.1. Metabolic profile of phenols in Lonicerae japonicae flos fermented broth The differential metabolites of phenols in the L. japonicae flos fermented broth mainly included 4 kinds of methoxyphenols and 7 kinds of phenolic glycosides (Fig. 5 ). The levels of methoxyphenols, including coniferyl aldehyde and eugenol, showed a significantly decreased after fermentation in all groups. The decrease in those levels of methoxyphenols could be attributed to the elevation of homovanillic acid, as coniferyl aldehyde and eugenol could be converted into homovanillic acid through the production of ferulic acid. The level of vanillin exhibited a decline in the SY group, whereas it was increased in the Picp-2 and Picp-2 + SY groups. Since the vanillin could be transformed into homovanillic acid by the action of oxidation. Homovanillic acid serves as a pivotal metabolite of dopamine, reflecting the neurotransmitter's turnover in the central nervous system. It is quantified in cerebrospinal fluid to evaluate the status of dopaminergic pathways, which is vital for diagnosing and monitoring various neurological conditions, including Parkinson's disease and neuroblastoma ( https://doi.org/10.1016/j.clinbiochem.2008.08.077 ). Moreover, homovanillic acid has been identified as a potential antioxidant, suggesting its role in combating oxidative stress associated with neurodegenerative diseases ( https://doi.org/10.1016/j.cmet.2024.03.010 ). In addition, L. plantarum Picp-2 had a conversion effect on phenolic glycosides. For example, the pentamidine (phenolic glycosides) was increased in both the Picp-2 and Picp-2 + SY groups. On the contrary, the levels of 5 kinds of phenolic glycosides were declined, such as coniferin. The decrease in coniferin levels could be attributed to the increase in ferulic acid, as coniferin can be transformed into coniferyl aldehyde and further synthesized into ferulic acid through oxidation. A substantial proportion of phenolic substances in plants exist in the form of aglycones, which require prior hydrolysis to remove glycosides before they can exert their effects. Microorganisms can directly hydrolyze the glycosidic bonds through fermentation, thereby facilitating the absorption and utilization of phenolic compounds by the human body. The hydrolysis not only improves the bioavailability but also potentially increases the antioxidant capacity and biological activity of these phenolic substances. The L. japonicae flos was a bountiful source of phenolic acids, with over 40 distinct phenolic acids had been isolated to date. The phenolic acids mainly include chlorogenic acid, cinnamic acid, coumaric acid and benzoic coumaric acid. In this study, there were more than 49 kinds of phenolic acids in L. japonicae flos fermented broth showed a differential expression after fermentation, including cinnamic acid, quinic acid and their analogues. Among them, the cinnamic acids (16 kinds) were the most, mainly including the increased levels of caffeic acid, caffeoylquinic acid, cinnamic acid, ferulic acid. Other phenolic acids including vanillic acid and protocatechuic acid were also showed an increase after fermentation. The phenolic compounds in plants exists in a bound form, complexed with cellular structures. Fermentation processes can convert these bound phenolic acids into their free forms, and through microbial metabolism, these free phenolic acids can be transformed into compounds with higher bioactivity. This conversion not only enhances the bioavailability of phenolic compounds but also potentially increases their antioxidant properties and other health benefits ( https://doi.org/10.1016/j.biotechadv.2021.107763 ). 3.5.2. Metabolic profile of flavonoids in Lonicerae japonicae flos fermented broth In this study, 50 kinds of differentially expressed flavonoid had been isolated from L. japonicae flos fermented broth, mainly included flavonols (eg. isorhamnetin, quercetin, kaempferol), flavones (eg. tricetin, 5-hydroxyflavone, baicalein, luteolin, apigenin) and isoflavones, and most of them were glycosides and methylated (Fig. 6 ). Other flavonoids included 20 kinds of methylated flavonoids, 4 kinds of biflavonoids and 12 kinds of flavonoid glycosides also had been identified from L. japonicae flos fermented broth. The degradations of flavonoid glycosides such as kaempferol-4'-glucoside, isoquercitrin, naringin, naringenin-7- O -glucoside were observed in the Picp-2 group. The levels of glabridin, irigenin, isorhamnetin were significantly decreased in the Picp-2 and Picp-2 + SY groups, while the eriodictyol, pinocembrin had increased. The degradation of tricaffeoyl quinic acid was attributed to the feruloyl esterase, which increased the levels of quinic acid and the corresponding hydroxycinnamic acid (ferulic acids, caffeic acids and p -coumaric acids). Indeed, concomitant production of metabolites derived from feruloyl esterase activity was notable in this study, such as dihydroferulic acid and ferulic acid. The release of quinic acid (3,5-di-caffeoylquinic acid and coumaroyl quinic acid) was only detected in Picp-2 and Picp-2 + SY groups, while tricaffeoyl quinic acid showed a significant decrease in Picp-2 and SY groups. Besides, there was a significant decrease in quinic acid and derivatives (3- O -feruloylquinic acid and 3- O -caffeoylquinic acid methyl ester) in Picp-2 group. The release of free hydroxycinnamic acids was advantageous because they possessed greater antioxidant activities and was more bioavailable than the conjugated ester form [ 31 ]. In the same manner, feruloyl esterase activity demonstrated by L. plantarum Picp-2 may improve antioxidant capacities and nutrient bioavailability in L. japonicae flos via the release of hydroxycinnamic acids. The liberation of free hydroxycinnamic acids is advantageous as these compounds exhibit greater antioxidant activities and are more bioavailable than their conjugated ester forms. This enhancement in bioavailability and antioxidant capacity, attributed to the feruloyl esterase activity demonstrated by L. plantarum Picp-2, signifies a significant advancement in maximizing the nutritional and therapeutic potential of L. japonicae flos. It demonstrates how microbial action can transform bound phenolic acids into more bioavailable forms, thereby enhancing their health benefits. 3.5.3. Metabolic profile of amino acids and polypeptides in Lonicerae japonicae flos fermented broth The levels of polypeptidesd, such as glutamylleucine, leucylproline, threonylleucine, Gly-Leu and Leu-Leu-Tyr, were observed to decrease. This indicated that they were hydrolysed by proteinases and peptidases derived from L. plantarum Picp-2 and S. cerevisiae SY (Fig. 7 ). Indeed, proteolysis of di- and tripeptides would explain the increases of L-leucine, threonine in Picp-2 and Picp-2 + SY groups, while the increase was not observed in SY group. The L-leucine and threonine were essential amino acid and the more studied branched-chain amino acids, which cannot synthesize autonomously by animals. Moreover, the L-leucine and threonine could promote protein synthesis through the mammalian target of rapamycin (mTOR) pathway in skeletal muscle [ 32 ]. Similarly, the alpha amino acid and their derivatives (eg. Ornithine, Glutamine, L-Pipecolic acid) also showed a significant increase in Picp-2 and Picp-2 + SY groups, and the increase was not observed in SY group. Microorganisms could metabolize proteins into smaller peptides and amino acids during fermentation, which are more readily absorbed and possess higher nutritional value. This biotransformation not only enhances the nutritional profile of the substrate but also improves its digestibility ( https://doi.org/10.3390/fermentation7020063 ). 3.5.4. Metabolic profile of sugars and organic acids in Lonicerae japonicae flos fermented broth In this study, 3 mono- and disaccharides and 2 hexoses were consumed by L. plantarum Picp-2 and S. cerevisiae SY. On the contrary, melibiose was synthesized in all fermentation group, and the increase of d-raffinose and was observed in SY and Picp-2 + SY groups. Additionally, the increase of melezitose was only detected in SY group (Fig. 8 ). Microorganisms were known to transform sugars into acids through tricarboxylic acid (TCA) cycle. Metabolic changes in sugars and organic acids reflected the functionality of glycolysis and the TCA cycle respectively, although it is worth noting that the TCA cycle was incomplete in lactobacilli. The malic acid and succinic acid were consumed and transformed into fumaric acid in Picp-2 group. The same findings were shown in orange juices fermented with L. plantarum and L. brevis for lactic and citric acids [ 33 ]. 2-Isopropylmalic acid was produced in large amounts in Picp-2, SY, Picp-2 + SY groups (9.10-, 5.30- and 9.74-fold increased, respectively). The 2-isopropyl-malic acid may be therapeutically beneficial in L. japonicae flos attributable to its mild DPPH antioxidant scavenging active, in addition to weak anti-bacterial activities against several foodborne pathogens [ 34 ]. 3.5.5. Metabolic profile of saponins in Lonicerae japonicae flos fermented broth Most of saponins from L. japonicae flos belonged to the oleanane type and hederagenin type. In this study, about 12 saponins were isolated, mainly consisted of steroidal saponins and triterpene saponins (Fig. 9 ). The steroidal saponins (α-solanine, α-chaconine, pseudojervine, edpetiline and ophiopogonin D) were significantly increased in Picp-2 and Picp-2 + SY groups, and were not detected in SY group. On the contrary, the enhancement of the levels of triterpene saponins were only observed in SY and Picp-2 + SY groups, such as saikosaponin A and O-glucopyranosylepiederagenin. 3.6. Metabolic pathway analysis The Fig. 10 mainly demonstrated the top 25 pathways mainly involved in the differential metabolites. During the fermentation with Picp-2, there were mainly included phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis, D-glutamine and D-glutamate metabolism, valine, leucine and isoleucine biosynthesis. During the fermentation with SY, in addition to the pathway enriched in Picp-2 fermentation, there was also a separate enrichment of sphingolipid metabolism and galactose metabolism. The metabolic pathway in the Picp-2 + SY fermentation was similar to that of the single strain fermentation, while there were different amounts of enriched metabolites in some pathway, such as arginine biosynthesis. Besides, the galactose metabolism in Picp-2 + SY fermentation showed a lower enrichment level than that of SY fermentation. The decrease of count of SY during co-fermentation could potentially be attributed to the limiting effect of Picp-2 on galactose metabolism. However, the preliminary metabolic profiles could not explain the underlying mechanisms of synthesis or degradation of the different metabolites. 3.7. Biotransformation pathway analysis of metabolite in Lonicerae japonicae flos fermented broth After the metabolome analysis of differential metabolite, two potential biotransformation pathways were detected during the fermentation of L. japonicae flos by L. plantarum Picp-2 and S. cerevisiae SY. The details were shown as followed: The L. plantarum Picp-2 has a stronger conversion ability to flavonoids and organic acid, as the levels of these compositions were significantly increased after fermentation, such as chlorogenic acid and luteolin. Phenylalanine, p -cinnamic acid, p -coumaric acid and p -coumaroyl-CoA are the important compositions in the shikimic acid pathway for the synthesis of chlorogenic acid. Phenylalanine could be transformed into p -cinnamic acid by the action of phenylalanine ammonia lyase, then added a hydroxyl group to site 4 under the effect of cynnamate 4-hydroxylase to synthesize p -coumaric acid. The p -coumaric acid was consumed and metabolized further to turn into p -coumaroyl quinic acid. In the end, a hydroxyl group was added to p -coumaroyl quinic acid at site 3, and converted to chlorogenic acid. In addition, the chlorogenic acid could be transformed into eriodictyol, and further converted into luteolin and taxifolin by the action of flavone synthase and flavanone 3-hydroxylase, respectively by L. plantarum Picp-2 (Fig. 11 A). Figure 11 B showed that eugenol and coniferin were transformed into coniferin alcohol by the action of eugenol hydroxlylase and glycosidase, respectively. Coniferin alcohol could convert into coniferin aldehyde, and further metabolized to produce ferulic acid. Besides, the increases of ferulic acid in Picp-2 and Picp-2 + SY groups could be explained by the declines of hydroxyferulic acid and p -coumaric acid, as hydroxyferulic acid and p -coumaric acid could be transformed into ferulic acid by the action of dehydroxylase and demethoxylase, respectively. Ferulic acid could be converted to caffeic acid and feruloyl-CoA by demethylase and ferulic acid Co-A ligase, respectively. Caffeic acid was consumed to convert into protocatechuic acid. Besides, feruloyl-CoA could also be transformed into protocatechuic acid. Vanillin was transformed into vanillic acid, and then into protocatechuic acid, by the action of vanillin dehydrogenase and vanillate- O -demethylase, respectively. In addition, vanillin could also be converted into homovanillic acid by oxidizing the aldehyde group to a carboxyl group. The S. cerevisiae SY had a more remarkable conversion effect on protocatechuic acid, as in the SY group, the level of protocatechuic acid was significantly decreased, and the level of 4-hydroxybenzoic acid and 4-hydroxybenzoicdehyde was higher than that of Picp-2 and Picp-2 + SY groups. 3.8. Correlation analysis 3.8.1. Correlation analysis between antioxidants activities and the contents of total phenols, flavonoids and differential metabolites In previous studies, conflicting results had been reported regarding the correlation between phenols content and antioxidative activities. Fang et al. found that the total phenolics and flavonoids contents of citrus fruits was positively correlated with the ABTS ·+ and DPPH· scavenging rate [ 35 ]. On the other hand, another study indicated that the polyphenols and flavonoids contents were weakly positively correlated with the antioxidative activities of avocado peelings [ 36 ]. In this study, the antioxidant activities of fermented L. japonicae flos were improved, while the contents of phenols and flavonoids were significantly decreased. Meanwhile, the correlation analysis indicated that the total phenols and flavonoids contents had no significant positive correlation with the increase of antioxidant activities. This result was similar to previous research, the content of phenols was negatively correlated with antioxidant activities in pretreated Cannabis sativa L. by Ganoderma lucidum , while the enhancement of antioxidant activities was owed to the increased levels of licoricidin and p -coumaric acid after pretreatment [ 37 ], while the licoricidin and p -coumaric acid also had a increase in this study. In present study, the correlation analysis indicated that the increase of DPPH· scavenging rate showed a significant positive correlation with the level of demethyltexasin ( R 2 = 0.9816, P < 0.05, Fig. 7 A). The demethyltexasin was a trihydroxyisoflavone, the -OH group on site 6 (A-ring) in demethyltexasin acted as a primary radical scavenging site [ 38 ]. Moreover, demethyltexasin had a significant hyaluronic acid inhibition rate, due to the hydroxyl group on site 6 and 7 of the isoflavone skeleton [ 39 ]. Besides, our result indicated that the increase of ·OH scavenging rate was positively correlated with the levels of ferulic acid ( R 2 = 0.9848, P < 0.05), chlorogenic acid ( R 2 = 0.9648, P < 0.05) and eriodictyol ( R 2 = 0.9986, P < 0.05) (Fig. 7 B C D). The ferulic acid and chlorogenic acid as common phenolic acids, exhibited potent antioxidant activities that were dependent on their chemical construction, such as the ring substituents, degree of methoxylation and the number of -OH groups [ 40 ]. Eriodictyol was a flavonoid that belongs to a subclass of flavanones, it had the potential to enhance cell protection through increased antioxidant activity [ 41 ]. In an in vivo study, eriodictyol showed decreased oxidative damage in human retinal pigment epithelial-19 cells by Nrf2, and the activation of its downstream shielding phase-II enzymes, namely, heme oxygenase 1 (HO-1) and NADPH quinone dehydrogenase 1 (NQO1) [ 41 ]. 3.8.2. Correlation analysis between cytoprotection activity and differential metabolites In this study, the correlation analysis found that the taxifolin ( R 2 = 0.9705, P < 0.05), paeonol ( R 2 = 0.9533, P < 0.05) and riboflavin ( R 2 = 0.9924, P < 0.05) (Fig. 12 E F G) were positively correlated with cytoprotection activity (Fig. 12 ). The cytoprotective effect of taxifolin was investigated in previous research [ 42 , 43 ]. Taxifolin could suppress the activity of cathepsin D, B, PARP, caspase 3, -7, and cell cycle-related proteins and by activating the hsp27 to protect HaCaT cells against damage [ 42 ]. The cytoprotective effect of paeonol has not been studied well. However, paeonol could alleviate renal injury in mice by regulating Nrf2 (NF-E2 p45-related factor 2) and NF-κB pathways [ 44 ], due to transcription factor Nrf2 regulates the cellular redox homoeostasis and cytoprotective responses [45]. 4. Conclusion Fermentation with L. plantarum Picp-2 and S. cerevisiae SY was an effective approach to improve the antioxidant and cytoprotection activities of L. japonicae flos. Metabolomic and correlation analysis have indicated that the enhancement of antioxidant activities was attributed to demethyltexasin, ferulic acid, chlorogenic acid, eriodictyol. Besides, the increases of cytoprotection activity was positively correlated with taxifolin, paeonol and riboflavin. Meanwhile, this study demonstrated that S. cerevisiae facilitated the growth of L. plantarum during co-fermentation,it could enhance the metabolic activity of L. plantarum , and promoting their fermentative action on L. japonicae flos, improving the efficiency of fermentation. The untargeted metabolomics indicated that the L. plantarum could increase the levels of steroidal saponins, amino acids, and alkaloids, while it could decrease the levels of quinic acid and terpene glycosides. The S. cerevisiae could increase level of triterpenoid saponins. The combination of L. plantarum and S. cerevisiae exhibited a similar metabolic change to those of the individual strain fermentation groups, including the increase in the levels of benzoic acids, coumaric acids, cinnamic acids, flavonoids and amino acids, however, the magnitude of these changes was relatively small. Additionally, metabolomic analysis results indicate that during the fermentation process, phenolic glycosides are hydrolyzed and transformed into phenolic compounds with higher activity. This study represents the first attempt to uncover the biotransformation pathways during the fermentation process of L. japonicae flos through metabolomic profiling, and identifying two potential conversion pathways. This investigation provides a molecular-level analysis of the metabolic changes in L. japonicae flos during fermentation and elucidates the reasons behind the enhancement of efficacy, provides a reference for obtaining L. japonicae flos and other plant-based co-fermented products with enhanced antioxidant and cell protective activities. Declarations Consent for publication 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, and agree to publish this article. Funding This work was supported by the the Young Scientists of Hunan Province (2022RC1151), China Agriculture Research System for Bast and Leaf Fiber Crops (CARS-16-E25), the Training Program for Excellent Young Innovators of Changsha (KQ2106095) and Agricultural Science and Technology Innovation Program of China (ASTIP-IBFC-06). 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1","display":"","copyAsset":false,"role":"figure","size":342240,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of pH and the contents of active composition in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. (A) Changes of pH during \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos fermentation. (B) Changes of contents of phenols, flavonoids in \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. (C) Changes of contents of reducing sugars, proteins in \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. Columns with different letters indicated significant statistical differences (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/0c184f2b4b9f3476c89ce96f.png"},{"id":92435640,"identity":"72175124-d04c-450d-98e2-bfbf95ca7c17","added_by":"auto","created_at":"2025-09-29 16:58:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":471339,"visible":true,"origin":"","legend":"\u003cp\u003eThe antioxidant and cytoprotection activities in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. (A) Changes of the ·OH, DPPH·, ABTS\u003csup\u003e·+\u003c/sup\u003e scavenging rate, and the FRAP of \u003cem\u003eLonicerae japonicae \u003c/em\u003eflos. (B) Changes in the effect of \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos on human keratinocytes' cell viability after being damaged by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Columns with different letters indicated significant statistical differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/3760bdc6f58530cf2dadd24c.png"},{"id":92436660,"identity":"efde67a6-9618-43e6-b25a-1e842ca4bfaf","added_by":"auto","created_at":"2025-09-29 17:14:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112667,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal compositions analysis (PCA) score plots for the unfermented (CK) and the fermentation with Picp-2, SY, Picp-2+SY in \u003cem\u003eLonicerae japonicae\u003c/em\u003eflos.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/0fd7ced77180d93816df7273.png"},{"id":92436661,"identity":"492ba80b-4944-482f-af07-3cc7d0f1a05f","added_by":"auto","created_at":"2025-09-29 17:14:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":289663,"visible":true,"origin":"","legend":"\u003cp\u003eThe volcano plots of differential metabolites of \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos during fermentation. (A) The differential metabolites during the fermentation with Picp-2 in \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. (B) The differential metabolites during the fermentation with SY in \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. (C) The differential metabolites during the fermentation with Picp-2+SY in \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/afd12cdc5cf9c53aefae3ad0.png"},{"id":92435645,"identity":"883f4e67-3c65-4444-a801-32d8dc55eae9","added_by":"auto","created_at":"2025-09-29 16:58:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2429600,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of differential metabolites of phenols in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/0f320e5866a62e9cd3186fbd.png"},{"id":92435936,"identity":"fc9aacef-89e3-48ea-94c0-fefa148d0fc5","added_by":"auto","created_at":"2025-09-29 17:06:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3264157,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of differential metabolites of flavonoids in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/a8658e067560ed960772a0d2.png"},{"id":92436662,"identity":"12777a41-3887-438f-a9f8-1fba086c07f1","added_by":"auto","created_at":"2025-09-29 17:14:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2424751,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of differential metabolites of amino acids and polypeptides in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003eflos.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/9971633511ed657ec0259ee4.png"},{"id":92435652,"identity":"01a7089a-3156-44ab-a036-b8896d5d261c","added_by":"auto","created_at":"2025-09-29 16:58:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1802037,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of differential metabolites of sugars and organic acids in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003eflos.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/7c95640f5db3fdad32ba8fc9.png"},{"id":92436664,"identity":"39201616-b363-4817-83d5-3c15298f600e","added_by":"auto","created_at":"2025-09-29 17:14:13","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2075882,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of differential metabolites of saponins in unfermented (CK) and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/57b6f10db658defa9e174609.png"},{"id":92436839,"identity":"b7889b67-a014-4fa4-88cc-9dec4992fdb7","added_by":"auto","created_at":"2025-09-29 17:22:13","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":448249,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of KEGG enrichment factor map of in unfermented and fermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. (A) Representation of KEGG enrichment factor map of differential metabolites in Picp-2 fermentation group; (B) Representation of KEGG enrichment factor map of differential metabolites in SY fermentation group; (C) Representation of KEGG enrichment factor map of differential metabolites in Picp-2+SY fermentation group.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/4d0d11fc793d62f687d13a5c.png"},{"id":92435658,"identity":"5b74c1c9-e2fa-4c28-b987-9d85236d503e","added_by":"auto","created_at":"2025-09-29 16:58:13","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":511458,"visible":true,"origin":"","legend":"\u003cp\u003ePotential biotransformation pathway of \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos fermentation. The frame represented the intensity of metabolites in each group, with dark red and blue indicating high and low peak intensities respectively. CK: unfermented \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. Picp-2: \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e fermented group,\u003cem\u003e \u003c/em\u003eSY: \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e fermented group. Picp-2+SY: Composite strain fermented group.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/db4c81ac170e5c71ea8fdc12.png"},{"id":92435665,"identity":"158415c3-5b12-4493-b45d-bd35c04db498","added_by":"auto","created_at":"2025-09-29 16:58:13","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":274090,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between antioxidants, cytoprotection\u003cem\u003e \u003c/em\u003eactivities and the metabolites. (A) The correlation analysis between DPPH· scavenging rate and the expression levels of demethyltexasin. The correlation analysis between ·OH scavenging rate and the expression levels of ferulic acid (B), chlorogenic acid (C), eriodictyol (D). The The correlation analysis between cytoprotection\u003cem\u003e \u003c/em\u003eactivity and the expression levels of taxifolin (E), paeonol (F), riboflavin(G).\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/eb3f52e925c6f023779c60c8.png"},{"id":92437527,"identity":"6a99e097-90a3-4090-b4a1-120a01515a00","added_by":"auto","created_at":"2025-09-29 17:30:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16005589,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7563014/v1/4ad622df-84da-46a5-a46f-6124d8747990.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Enhancement of Antioxidant and Cytoprotective Activities in Lonicerae Japonicae Flos via Co-fermentation: Metabolomics Unraveling Key Biotransformation Pathways","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eLonicerae japonicae\u003c/em\u003e flos was the dried bud or flower of \u003cem\u003eLonicera japonica\u003c/em\u003e Thunb. It was a traditional medicine material that belongs to the category of medicine food homology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Modern pharmacological researches had demonstrated that the extracts from \u003cem\u003eL. japonicae\u003c/em\u003e flos possessed a diverse range of biological activities, such as antioxidant, antimicrobial, antitumor, antiviral, and immunomodulatory effects [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The \u003cem\u003eL. japonicae\u003c/em\u003e flos was also used to prevent COVID-19, on account of the inhibition of SARS-CoV-2 M\u003csup\u003epro\u003c/sup\u003e activity, thereby alleviating viral entry as well as replication [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In previous reports, over 200 active compounds had been isolated from \u003cem\u003eL. japonicae\u003c/em\u003e flos, including phenols, organic acids, flavonoids, monoterpenoids, triterpenoids, volatile oils and lignans [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, these active compounds were typically bound to cellular structures, such as hemicellulose, xylan, cellulose and lignin. Therefore, the extraction methods of active compounds in \u003cem\u003eL. japonicae\u003c/em\u003e flos, including water extraction, ultrasonic alcohol extraction and enzyme-assisted extraction, had a limited extraction efficiency [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFermentation was a common biotransformation method, which could liberate active compounds bound to the cellular structures and improved their extraction efficiency [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It was reported that after the co-fermentation of lactobacillus and yeast, the phenols and flavonoids of brown rice increased by 93.3% and 61.3%, respectively [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, fermentation could leverage the metabolic activities of microorganisms to convert active compositions in the substrate, augment its original efficacy and generate novel effects. For example, the value of FRAP and DPPH\u0026middot; scavenging rate of fruit juice fermented by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus salivarius\u003c/em\u003e, and \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e were significantly increased compared with the control group [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, fermentation could enhance the scope of application and utilization of plant materials, and develop more valuable products. However, only a few studies had been focused on the fermentation of \u003cem\u003eL. japonicae\u003c/em\u003e flos, let alone on the metabolites changes during the fermentation.\u003c/p\u003e\u003cp\u003eUntargeted metabolomics based on LC-MS provided a comprehensive overview of all known and unknown metabolites, thereby justifying the potential active compounds biotransformation during fermentation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Li et al. utilized the untargeted metabolomics to isolate 66 volatile compounds and 30 nonvolatile compounds of \u003cem\u003eL. japonicae\u003c/em\u003e flos processed with different drying methods [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In another study, metabolomics had found 27, 81, 113 differential metabolites in flowers, flower buds, leaves of \u003cem\u003eL. japonicae\u003c/em\u003e Thunb [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Overall, untargeted metabolomics based on LC-MS proved to be an effective approach for elucidating the metabolic changes during \u003cem\u003eL. japonicae\u003c/em\u003e flos fermentation.\u003c/p\u003e\u003cp\u003eAbove all, this study aim to improve the extraction efficiency of active substances in \u003cem\u003eL. japonicae\u003c/em\u003e flos by using a novel biopretreatment method as well as improve the antioxidant and cytoprotection activities of \u003cem\u003eL. japonicae\u003c/em\u003e flos after fermentation, and metabolomics is also used to reveal the reasons for the changes in substances and activities at the molecular level. This study could provide a reference for the biotransformation of metabolites in \u003cem\u003eL. japonicae\u003c/em\u003e flos, facilitating the development of new products with improved functionality fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals and standards\u003c/h2\u003e\u003cp\u003e1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2\u0026acute;-azino-bis (3-ethylb-enzothiazoline-6-sul-fonic acid) (ABTS), Folin-Ciocalteu reagent, gallic acid were purchased from Sigma Chemicals Co. (St Louis, MO, USA). 3,5-dinitrosalicylic acid, ascorbic acid, gallic acid, rutin, coomassie brilliant blue, vitamin E, D-glucose were purchased fromo Merk (Darmstadt, Germany). Celluclast\u0026reg; (700 U/g) was purchased from Novozymes (China) Biotechnology Co., Ltd (Tianjin, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Samples and microorganism collection\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLonicerae japonicae\u003c/em\u003e flos was purchased from Hunan Longhui Yi-du Se-enriched Tea Industry Co., Ltd. (Hunan, China). \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e Picp-2 (CCTCC M20191045) was purchased from the China Center for Type Culture Collection (Wuhan, China). \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e SY (CGMCC 2.119) was purchased from China General Microbial Culture Preservation and Management Center (Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Sample processing\u003c/h2\u003e\u003cp\u003eTen grams of \u003cem\u003eL. japonicae\u003c/em\u003e flos powder (crushed and filtered through a 60-mesh sieve) were mixed with 200 mL of 630 U/mL cellulase solution (0.45%, w/v). Then the mixture was put into a constant temperature oscillation box at 50℃ and stirred for 6 h at 180 rpm. The mixed enzymatic hydrolysate of the \u003cem\u003eL. japonicae\u003c/em\u003e flos was prepared for further fermentation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of fermented Lonicerae japonicae flos\u003c/h2\u003e\u003cp\u003eThe enzymatic hydrolysate of the \u003cem\u003eL. japonicae\u003c/em\u003e flos (as described in 2.3) was sterilized at 121℃ for 15 min. The cells were then inoculated into 250 mL conical flasks containing 200 mL enzymatic hydrolysate of \u003cem\u003eL. japonicae\u003c/em\u003e flos with a population of 2% \u003cem\u003eS. cerevisiae\u003c/em\u003e SY and 10% \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2, in both single and co-culture groups, then incubated at 37℃ for 72 h [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The initial microbial populations were established based on previous researches conducted on fermented beverages. After fermentation, they were centrifuged twice (7000 rpm, 4℃, 5 min) and the supernatant was gathered and stored at -20℃ for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Determination of antioxidant activities\u003c/h2\u003e\u003cp\u003eIn vitro antioxidant activities of fermented broth were evaluated in terms of the scavenging rate of 1,1-diphenyl-2-picryl-hydrazyl radicals (DPPH\u0026middot;), 2,2\u0026acute;-azino-bis (3-ethylb-enzothiazoline-6-sulfonic acid) radicals (ABTS\u003csup\u003e\u0026middot;+\u003c/sup\u003e), hydroxyl radicals (\u0026middot;OH) and ferric reducing antioxidant power (FRAP) assay as described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the ascorbic acid (0.8 mg/mL) was used as a positive control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Determination of active compositions content\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1. Determination of total phenols content\u003c/h2\u003e\u003cp\u003eThe total phenols content in the fermented broth was quantified using Folin-Ciocalteu reagents with gallic acid as the reference standard. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. One milliliter of fermented broth was diluted by a factor of 10 and mixed with 1.5 mL of Folin-Ciocalteu reagent for 3\u0026ndash;8 minutes at room temperature. Then 1 mL of 20% (w/v) Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e was added to the mixtures and adjusted final total volume to 10 mL with 6.5 mL of dH\u003csub\u003e2\u003c/sub\u003eO. The mixtures were incubated for 1 h to facilitate the chromogenic reaction. Afterwards, their optical density was determined at a wavelength of 760 nm. The total phenols content was calculated according to the gallic acid calibration curve (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9959) and expressed as mg gallic acid equivalents (GAE)/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2. Determination of total flavonoids content\u003c/h2\u003e\u003cp\u003eThe total content of flavonoids in the fermented broth was quantified using a colorimetric method with rutin as the reference standard [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. One milliliter fermented broth was mixed with 12 mL of 75% (v/v) ethanol and 1 mL of 5% (w/w) NaNO\u003csub\u003e2\u003c/sub\u003e for a duration of 5 min, followed by the addition of 1 mL of 10% (w/w) Al(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e. After a 5-minute incubation period, 10 mL of NaOH solution (1 M) was introduced into the mixture. The solution was reacted for 15 min. Afterwards, the absorbance of the mixture was measured at 510 nm. The total flavonoid content was calculated from the calibration curve of rutin standard solution (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9912) and expressed as mg rutin equivalents (RTE)/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3. Determination of reducing sugars content\u003c/h2\u003e\u003cp\u003eThe quantification of reducing sugars were performed using the 3,5-dinitrosalicylic acid (DNS) method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The DNS reagent was prepared by dissolving 1 g of DNS and 30 g of potassium sodium tartrate in 80 mL of 0.5 M NaOH at a temperature of 45℃ with vigorous stirring until complete dissolution. After cooling to room temperature, the solution was diluted with ddH\u003csub\u003e2\u003c/sub\u003eO to a final volume of 100 mL. For the measurement, 2 mL of DNS reagent were added to a tube containing 1 mL of fermented broth and incubated at 95℃ for 5 minutes. After cooling, the solution was diluted with 7 mL of distilled water and the absorbance was measured at 540 nm using a microplate reader. The reducing sugar content was determined using a calibration curve of standard glucose (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9975) and expressed as mg glucose equivalent (GE)/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4. Determination of total proteins content\u003c/h2\u003e\u003cp\u003eThe quantification of total proteins were performed using the Bradford method. Twenty \u0026micro;L of fermented broth were mixed with 200 \u0026micro;L of coomassie brilliant blue (CBB). After 10 min, the absorbance of the mixture was measured at 595 nm in a microplate reader. The protein concentrations were determined according to the absorbance rate of the sample and a 300 mg/L bovine albumin.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Cytoprotection activity of fermented Lonicerae japonicae flos\u003c/h2\u003e\u003cp\u003eTo evaluate the protective efficacy of fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos against oxidative stress induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], human keratinocytes were pretreated with unfermented and fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos for 30 min. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was then added (0.5 mmol/L in HaCaT) for 4 h. For each experiment, the controls were prepared using the same concentration of DMSO as the samples and containing an appropriate volume of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/sterile water along with vitamin E. (300 \u0026micro;g/mL) was used as a positive control. After treatment, the protective effect of fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos was estimated by neutral red uptake assay.\u003c/p\u003e\u003cp\u003eNeutral red (NR) at a concentration of 0.03% (m/v) in phosphate-buffered saline (PBS) was introduced to the cells. The human keratinocytes were incubated for an additional 2 hours at 37℃, then washed with a formaldehyde (0.125%, v/v) and CaCl\u003csub\u003e2\u003c/sub\u003e (0.25%, m/v) mixture before dissolving NR retained in the cells using acetic acid (1%, v/v) in methanol (50%, v/v). The plates were read on a microplate reader at 540 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Untargeted metabolomics analysis\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1. Preparation of fermented Lonicerae japonicae flos metabolome samples\u003c/h2\u003e\u003cp\u003eThe fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos was freeze dried in a lyophilizer. The sample was subsequently dissolved in a mixture of acetonitrile and methanol (8:2 v/v) at a solid-liquid ratio of 1:50 (w/v) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Samples were then centrifuged at 8000 rpm for 5 min. The supernatants were filtered through 0.22 \u0026micro;m filter membrane. Mixed all extracts to be a pooled sample as a quality control (QC) sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2. Metabolomics analysis by LC-MS\u003c/h2\u003e\u003cp\u003eConditions for ultra performance liquid chromatography (UHPLC) analysis involved the use of Agilent 1200 UHPLC system (Waldbronn, Germany) with a SunFireTM C18 chromatographic column (250 x 4.6 mm, 5 microns, Waters, USA). The metabolic compounds were detected by Q-Exactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) under two ion modes (positive-ion and negative-ion modes). The gradient elution procedure was conducted with eluent A (0.1% formic acid in water, v/v) and eluent B (Acetonitrile). The elution procedure was 0\u0026ndash;5 min, 15% B; 5\u0026ndash;10 min, 15\u0026ndash;20% B; 10\u0026ndash;20 min, 20\u0026ndash;25% B; 20\u0026ndash;30 min, 25\u0026ndash;35% B; 30\u0026ndash;40 min, 35\u0026ndash;50% B; 40\u0026ndash;50 min, 80% B; For 50\u0026ndash;55 minutes, 15% B. The flow rate was maintained at 0.3 mL/min. The column temperature was kept at 30 ℃, and the sample injection volume was set at 5 \u0026micro;L. Q-Exactive quadrupole-Orbitrap mass spectrometer was operated at capillary temperature of 320℃, sheath gas flow rate of 35 psi and aux gas flow rate of 10 L/min, aux gas heater temperature of 350℃. The scan range covered 50-1000 m/z in the negative-ion mode and 50-2000 m/z in the positive-ion mode.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Metabolomics data processing\u003c/h2\u003e\u003cp\u003eThe LC-MS/MS data were processed and analyzed using multivariate techniques, as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Principal component analysis (PCA) and t-test were used to identify the differential metabolites among the samples. Metabolites with variable importance in projection (VIP)\u0026thinsp;\u0026gt;\u0026thinsp;1.0 and a \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to be differential metabolites. The heatmaps were created on OeBiotech (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.com/task/\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.com/task/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll measurements were performed in triplicate. Results in the present study were shown as means. Statistical comparisons were made using a two-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as statistical significance. Columns with different letters indicate a significant statistical disparity. Correlation analysis was performed by using a two-tailed Pearson's correlation test of GraphPad Prism version 9.4 software package.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e3.1. The pH and cell count in Lonicerae japonicae\u003c/em\u003e flos \u003cem\u003efermented broth\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe utilization of a combination of lactobacilli and yeast for fermentation had been widely reported [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], with much publication dedicated to their interactions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], However, there are still no reports of adopting this fermentation strategy in \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos. In present study, compared with the unfermented group, the pH of Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups were decreased to 3.65\u0026ndash;3.89 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, the count of \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 in Picp-2\u0026thinsp;+\u0026thinsp;SY group (6.36 log CFU/mL) was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that in Picp-2 group (6.07 log CFU/mL). As was widely recognized, yeast could produce ethanol and CO\u003csub\u003e2\u003c/sub\u003e, which induced the stress response in lactobacilli, thereby enhancing thermal tolerance and promoting the biosynthesis of unsaturated fatty acids [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The CO\u003csub\u003e2\u003c/sub\u003e could also create an anaerobic environment that promote the growth of lactobacilli [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Meanwhile, yeast could metabolize and produce amino acids to promote the growth of lactobacilli [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As opposed to lactobacilli, the count of \u003cem\u003eS. cerevisiae\u003c/em\u003e SY in Picp-2\u0026thinsp;+\u0026thinsp;SY group was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that in SY group. Previous study showed that the lactobacilli could produce a mass of acid, which result in an extremely low pH environment, that effectively suppressed yeast growth [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This result indicated that \u003cem\u003eS. cerevisiae\u003c/em\u003e could promote the growth of \u003cem\u003eL. plantarum\u003c/em\u003e. Conversely, \u003cem\u003eL. plantarum\u003c/em\u003e inhibited the growth of \u003cem\u003eS. cerevisiae\u003c/em\u003e. Therefore, co-fermentation is beneficial to promote the metabolic activity of the single strain, so that it can more actively interact with the fermentation substrate and improve the extraction efficiency of active substances.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2. The contents of phenols, flavonoids, reducing sugars and proteins in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eCompared with the unfermented group, the total phenols content in the Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups reduced from 59.67 mg GAE/mL to 51.99 and 53.09 mg GAE/mL (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The changing of total flavonoids content in these groups was similar to that of total phenols content. In addition, the content of reducing sugars in Picp-2, SY and Picp-2\u0026thinsp;+\u0026thinsp;SY groups was reduced to 0.23\u0026ndash;0.29 mg GE/mL (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the total protein content in Picp-2, SY and Picp-2\u0026thinsp;+\u0026thinsp;SY groups was decreased to 0.18\u0026ndash;0.20 mg/mL (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). There was some controversy regarding the release of substances from substrates by fermentation. Some reports showed that the microorganism could promote the release of phenols and flavonoids of substrate, while in another study, microorganism could consume and reduce the phenols and flavonoids during fermentation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our result showed that fermentation with Picp-2 and SY could reduce the content of phenols and flavonoids in \u003cem\u003eL. japonicae\u003c/em\u003e flos.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Antioxidants and Cytoprotection activities in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eCompared with the unfermented group (69.02%), the \u0026middot;OH scavenging rate of the Picp-2 (85.06%) and Picp-2\u0026thinsp;+\u0026thinsp;SY (77.88%) groups showed a significant increase (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), while the SY group (65.28%) had not significant change (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The DPPH\u0026middot; scavenging rate of the SY group was significantly increased to 85.43%, nevertheless, the Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups did not exhibit significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The ABTS\u003csup\u003e\u0026middot;+\u003c/sup\u003e scavenging rate of fermtation groups had no significant differences after fermentation. The FRAP of the SY group (1.026) was significantly increased compared with the unfermented group (1.002). On the contrary, the FRAP of Picp-2 group (9.708) was significantly decreased after fermentation, and the Picp-2\u0026thinsp;+\u0026thinsp;SY group had not significantly changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Bsides, there was also an increase in cytoprotection of \u003cem\u003eL. japonicae\u003c/em\u003e flos, compared with the unfermented group, the cell viability of the Picp-2\u0026thinsp;+\u0026thinsp;SY group treated human keratinocytes significantly increased from 49.13% to 64.94%. However, the cell viability of Picp-2 (51.33%) and SY (53.53%) groups treated human keratinocytes had no significant difference compared with unfermented group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This result indicated that the fermentation with Picp-2 and SY could improve the antioxidant and cytoprotection activities of \u003cem\u003eL. japonicae\u003c/em\u003e flos. The most use of \u003cem\u003eL. japonicae\u003c/em\u003e flos is for drinking and has strict requirements for \"large white flower\" and \"two white flower\" buds, which limits its use. This study removed the limitation of flowering period and enhance the antioxidant and cytoprotective activities of \u003cem\u003eL. japonicae\u003c/em\u003e flos, which provides a broader market prospect for its development and application.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Metabolic profile in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eTo gain a comprehensive understanding of the changes of metabolites during \u003cem\u003eL. japonicae\u003c/em\u003e flos fermentation, the \u003cem\u003eL. japonicae\u003c/em\u003e flos fermented broth and the unfermented group were chosen for the following metabonomics analysis. A total of 1082 and 442 metabolites were identified in positive and negative ion mode, respectively, mainly including phenols, flavonoids, amino acids, peptides, sugars, organic acids and saponins. PCA analysis showed that the unfermented and fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos could separate well from each group and differential metabolites analysis of the metabonomics could be performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Differential metabolites analysis of Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eThe volcano plot could clearly depict the significant situation of differential expression of metabolites. A total of 576, 358, 651 differential metabolites was identified in Picp-2, SY, Picp-2\u0026thinsp;+\u0026thinsp;SY groups, respectively. Among them, 274, 119, 301 metabolites were up-regulated in Picp-2, SY, Picp-2\u0026thinsp;+\u0026thinsp;SY groups, along with 302, 239, 350 metabolites were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA B C). Compared with unfermented groups, the expression levels of benzoic acids, coumaric acids, cinnamic acids, fatty acids and flavonoids in the Picp-2 and SY groups were significantly increased. The exclusive increase of steroidal saponins, amino acids, alkaloids, quinic acids, terpene glycosides and catechin was observed in the Picp-2 group. The level of triterpenoid saponins was only increased in SY group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.5.1. Metabolic profile of phenols in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eThe differential metabolites of phenols in the \u003cem\u003eL. japonicae\u003c/em\u003e flos fermented broth mainly included 4 kinds of methoxyphenols and 7 kinds of phenolic glycosides (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The levels of methoxyphenols, including coniferyl aldehyde and eugenol, showed a significantly decreased after fermentation in all groups. The decrease in those levels of methoxyphenols could be attributed to the elevation of homovanillic acid, as coniferyl aldehyde and eugenol could be converted into homovanillic acid through the production of ferulic acid. The level of vanillin exhibited a decline in the SY group, whereas it was increased in the Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups. Since the vanillin could be transformed into homovanillic acid by the action of oxidation. Homovanillic acid serves as a pivotal metabolite of dopamine, reflecting the neurotransmitter's turnover in the central nervous system. It is quantified in cerebrospinal fluid to evaluate the status of dopaminergic pathways, which is vital for diagnosing and monitoring various neurological conditions, including Parkinson's disease and neuroblastoma (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.clinbiochem.2008.08.077\u003c/span\u003e\u003cspan address=\"10.1016/j.clinbiochem.2008.08.077\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Moreover, homovanillic acid has been identified as a potential antioxidant, suggesting its role in combating oxidative stress associated with neurodegenerative diseases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cmet.2024.03.010\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2024.03.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 had a conversion effect on phenolic glycosides. For example, the pentamidine (phenolic glycosides) was increased in both the Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups. On the contrary, the levels of 5 kinds of phenolic glycosides were declined, such as coniferin. The decrease in coniferin levels could be attributed to the increase in ferulic acid, as coniferin can be transformed into coniferyl aldehyde and further synthesized into ferulic acid through oxidation. A substantial proportion of phenolic substances in plants exist in the form of aglycones, which require prior hydrolysis to remove glycosides before they can exert their effects. Microorganisms can directly hydrolyze the glycosidic bonds through fermentation, thereby facilitating the absorption and utilization of phenolic compounds by the human body. The hydrolysis not only improves the bioavailability but also potentially increases the antioxidant capacity and biological activity of these phenolic substances.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eL. japonicae\u003c/em\u003e flos was a bountiful source of phenolic acids, with over 40 distinct phenolic acids had been isolated to date. The phenolic acids mainly include chlorogenic acid, cinnamic acid, coumaric acid and benzoic coumaric acid. In this study, there were more than 49 kinds of phenolic acids in \u003cem\u003eL. japonicae\u003c/em\u003e flos fermented broth showed a differential expression after fermentation, including cinnamic acid, quinic acid and their analogues. Among them, the cinnamic acids (16 kinds) were the most, mainly including the increased levels of caffeic acid, caffeoylquinic acid, cinnamic acid, ferulic acid. Other phenolic acids including vanillic acid and protocatechuic acid were also showed an increase after fermentation. The phenolic compounds in plants exists in a bound form, complexed with cellular structures. Fermentation processes can convert these bound phenolic acids into their free forms, and through microbial metabolism, these free phenolic acids can be transformed into compounds with higher bioactivity. This conversion not only enhances the bioavailability of phenolic compounds but also potentially increases their antioxidant properties and other health benefits (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2021.107763\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2021.107763\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.5.2. Metabolic profile of flavonoids in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eIn this study, 50 kinds of differentially expressed flavonoid had been isolated from \u003cem\u003eL. japonicae\u003c/em\u003e flos fermented broth, mainly included flavonols (eg. isorhamnetin, quercetin, kaempferol), flavones (eg. tricetin, 5-hydroxyflavone, baicalein, luteolin, apigenin) and isoflavones, and most of them were glycosides and methylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Other flavonoids included 20 kinds of methylated flavonoids, 4 kinds of biflavonoids and 12 kinds of flavonoid glycosides also had been identified from \u003cem\u003eL. japonicae\u003c/em\u003e flos fermented broth.\u003c/p\u003e\u003cp\u003eThe degradations of flavonoid glycosides such as kaempferol-4'-glucoside, isoquercitrin, naringin, naringenin-7-\u003cem\u003eO\u003c/em\u003e-glucoside were observed in the Picp-2 group. The levels of glabridin, irigenin, isorhamnetin were significantly decreased in the Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups, while the eriodictyol, pinocembrin had increased. The degradation of tricaffeoyl quinic acid was attributed to the feruloyl esterase, which increased the levels of quinic acid and the corresponding hydroxycinnamic acid (ferulic acids, caffeic acids and \u003cem\u003ep\u003c/em\u003e-coumaric acids). Indeed, concomitant production of metabolites derived from feruloyl esterase activity was notable in this study, such as dihydroferulic acid and ferulic acid. The release of quinic acid (3,5-di-caffeoylquinic acid and coumaroyl quinic acid) was only detected in Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups, while tricaffeoyl quinic acid showed a significant decrease in Picp-2 and SY groups. Besides, there was a significant decrease in quinic acid and derivatives (3-\u003cem\u003eO\u003c/em\u003e-feruloylquinic acid and 3-\u003cem\u003eO\u003c/em\u003e-caffeoylquinic acid methyl ester) in Picp-2 group. The release of free hydroxycinnamic acids was advantageous because they possessed greater antioxidant activities and was more bioavailable than the conjugated ester form [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the same manner, feruloyl esterase activity demonstrated by \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 may improve antioxidant capacities and nutrient bioavailability in \u003cem\u003eL. japonicae\u003c/em\u003e flos via the release of hydroxycinnamic acids. The liberation of free hydroxycinnamic acids is advantageous as these compounds exhibit greater antioxidant activities and are more bioavailable than their conjugated ester forms. This enhancement in bioavailability and antioxidant capacity, attributed to the feruloyl esterase activity demonstrated by \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2, signifies a significant advancement in maximizing the nutritional and therapeutic potential of \u003cem\u003eL. japonicae\u003c/em\u003e flos. It demonstrates how microbial action can transform bound phenolic acids into more bioavailable forms, thereby enhancing their health benefits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.5.3. Metabolic profile of amino acids and polypeptides in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eThe levels of polypeptidesd, such as glutamylleucine, leucylproline, threonylleucine, Gly-Leu and Leu-Leu-Tyr, were observed to decrease. This indicated that they were hydrolysed by proteinases and peptidases derived from \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 and \u003cem\u003eS. cerevisiae\u003c/em\u003e SY (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Indeed, proteolysis of di- and tripeptides would explain the increases of L-leucine, threonine in Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups, while the increase was not observed in SY group. The L-leucine and threonine were essential amino acid and the more studied branched-chain amino acids, which cannot synthesize autonomously by animals. Moreover, the L-leucine and threonine could promote protein synthesis through the mammalian target of rapamycin (mTOR) pathway in skeletal muscle [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, the alpha amino acid and their derivatives (eg. Ornithine, Glutamine, L-Pipecolic acid) also showed a significant increase in Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups, and the increase was not observed in SY group. Microorganisms could metabolize proteins into smaller peptides and amino acids during fermentation, which are more readily absorbed and possess higher nutritional value. This biotransformation not only enhances the nutritional profile of the substrate but also improves its digestibility (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/fermentation7020063\u003c/span\u003e\u003cspan address=\"10.3390/fermentation7020063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.5.4. Metabolic profile of sugars and organic acids in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eIn this study, 3 mono- and disaccharides and 2 hexoses were consumed by \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 and \u003cem\u003eS. cerevisiae\u003c/em\u003e SY. On the contrary, melibiose was synthesized in all fermentation group, and the increase of d-raffinose and was observed in SY and Picp-2\u0026thinsp;+\u0026thinsp;SY groups. Additionally, the increase of melezitose was only detected in SY group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Microorganisms were known to transform sugars into acids through tricarboxylic acid (TCA) cycle. Metabolic changes in sugars and organic acids reflected the functionality of glycolysis and the TCA cycle respectively, although it is worth noting that the TCA cycle was incomplete in lactobacilli. The malic acid and succinic acid were consumed and transformed into fumaric acid in Picp-2 group. The same findings were shown in orange juices fermented with \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. brevis\u003c/em\u003e for lactic and citric acids [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 2-Isopropylmalic acid was produced in large amounts in Picp-2, SY, Picp-2\u0026thinsp;+\u0026thinsp;SY groups (9.10-, 5.30- and 9.74-fold increased, respectively). The 2-isopropyl-malic acid may be therapeutically beneficial in \u003cem\u003eL. japonicae\u003c/em\u003e flos attributable to its mild DPPH antioxidant scavenging active, in addition to weak anti-bacterial activities against several foodborne pathogens [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e3.5.5. Metabolic profile of saponins in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eMost of saponins from \u003cem\u003eL. japonicae\u003c/em\u003e flos belonged to the oleanane type and hederagenin type. In this study, about 12 saponins were isolated, mainly consisted of steroidal saponins and triterpene saponins (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The steroidal saponins (α-solanine, α-chaconine, pseudojervine, edpetiline and ophiopogonin D) were significantly increased in Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups, and were not detected in SY group. On the contrary, the enhancement of the levels of triterpene saponins were only observed in SY and Picp-2\u0026thinsp;+\u0026thinsp;SY groups, such as saikosaponin A and O-glucopyranosylepiederagenin.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Metabolic pathway analysis\u003c/h2\u003e\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e mainly demonstrated the top 25 pathways mainly involved in the differential metabolites. During the fermentation with Picp-2, there were mainly included phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis, D-glutamine and D-glutamate metabolism, valine, leucine and isoleucine biosynthesis. During the fermentation with SY, in addition to the pathway enriched in Picp-2 fermentation, there was also a separate enrichment of sphingolipid metabolism and galactose metabolism. The metabolic pathway in the Picp-2\u0026thinsp;+\u0026thinsp;SY fermentation was similar to that of the single strain fermentation, while there were different amounts of enriched metabolites in some pathway, such as arginine biosynthesis. Besides, the galactose metabolism in Picp-2\u0026thinsp;+\u0026thinsp;SY fermentation showed a lower enrichment level than that of SY fermentation. The decrease of count of SY during co-fermentation could potentially be attributed to the limiting effect of Picp-2 on galactose metabolism. However, the preliminary metabolic profiles could not explain the underlying mechanisms of synthesis or degradation of the different metabolites.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Biotransformation pathway analysis of metabolite in Lonicerae japonicae flos fermented broth\u003c/h2\u003e\u003cp\u003eAfter the metabolome analysis of differential metabolite, two potential biotransformation pathways were detected during the fermentation of \u003cem\u003eL. japonicae\u003c/em\u003e flos by \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 and \u003cem\u003eS. cerevisiae\u003c/em\u003e SY. The details were shown as followed:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 has a stronger conversion ability to flavonoids and organic acid, as the levels of these compositions were significantly increased after fermentation, such as chlorogenic acid and luteolin. Phenylalanine, \u003cem\u003ep\u003c/em\u003e-cinnamic acid, \u003cem\u003ep\u003c/em\u003e-coumaric acid and \u003cem\u003ep\u003c/em\u003e-coumaroyl-CoA are the important compositions in the shikimic acid pathway for the synthesis of chlorogenic acid. Phenylalanine could be transformed into \u003cem\u003ep\u003c/em\u003e-cinnamic acid by the action of phenylalanine ammonia lyase, then added a hydroxyl group to site 4 under the effect of cynnamate 4-hydroxylase to synthesize \u003cem\u003ep\u003c/em\u003e-coumaric acid. The \u003cem\u003ep\u003c/em\u003e-coumaric acid was consumed and metabolized further to turn into \u003cem\u003ep\u003c/em\u003e-coumaroyl quinic acid. In the end, a hydroxyl group was added to \u003cem\u003ep\u003c/em\u003e-coumaroyl quinic acid at site 3, and converted to chlorogenic acid. In addition, the chlorogenic acid could be transformed into eriodictyol, and further converted into luteolin and taxifolin by the action of flavone synthase and flavanone 3-hydroxylase, respectively by \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB showed that eugenol and coniferin were transformed into coniferin alcohol by the action of eugenol hydroxlylase and glycosidase, respectively. Coniferin alcohol could convert into coniferin aldehyde, and further metabolized to produce ferulic acid. Besides, the increases of ferulic acid in Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups could be explained by the declines of hydroxyferulic acid and \u003cem\u003ep\u003c/em\u003e-coumaric acid, as hydroxyferulic acid and \u003cem\u003ep\u003c/em\u003e-coumaric acid could be transformed into ferulic acid by the action of dehydroxylase and demethoxylase, respectively. Ferulic acid could be converted to caffeic acid and feruloyl-CoA by demethylase and ferulic acid Co-A ligase, respectively. Caffeic acid was consumed to convert into protocatechuic acid. Besides, feruloyl-CoA could also be transformed into protocatechuic acid. Vanillin was transformed into vanillic acid, and then into protocatechuic acid, by the action of vanillin dehydrogenase and vanillate-\u003cem\u003eO\u003c/em\u003e-demethylase, respectively. In addition, vanillin could also be converted into homovanillic acid by oxidizing the aldehyde group to a carboxyl group. The \u003cem\u003eS. cerevisiae\u003c/em\u003e SY had a more remarkable conversion effect on protocatechuic acid, as in the SY group, the level of protocatechuic acid was significantly decreased, and the level of 4-hydroxybenzoic acid and 4-hydroxybenzoicdehyde was higher than that of Picp-2 and Picp-2\u0026thinsp;+\u0026thinsp;SY groups.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Correlation analysis\u003c/h2\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003e3.8.1. Correlation analysis between antioxidants activities and the contents of total phenols, flavonoids and differential metabolites\u003c/h2\u003e\u003cp\u003eIn previous studies, conflicting results had been reported regarding the correlation between phenols content and antioxidative activities. Fang et al. found that the total phenolics and flavonoids contents of citrus fruits was positively correlated with the ABTS\u003csup\u003e\u0026middot;+\u003c/sup\u003e and DPPH\u0026middot; scavenging rate [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. On the other hand, another study indicated that the polyphenols and flavonoids contents were weakly positively correlated with the antioxidative activities of avocado peelings [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, the antioxidant activities of fermented \u003cem\u003eL. japonicae\u003c/em\u003e flos were improved, while the contents of phenols and flavonoids were significantly decreased. Meanwhile, the correlation analysis indicated that the total phenols and flavonoids contents had no significant positive correlation with the increase of antioxidant activities. This result was similar to previous research, the content of phenols was negatively correlated with antioxidant activities in pretreated \u003cem\u003eCannabis sativa\u003c/em\u003e L. by \u003cem\u003eGanoderma lucidum\u003c/em\u003e, while the enhancement of antioxidant activities was owed to the increased levels of licoricidin and \u003cem\u003ep\u003c/em\u003e-coumaric acid after pretreatment [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], while the licoricidin and \u003cem\u003ep\u003c/em\u003e-coumaric acid also had a increase in this study.\u003c/p\u003e\u003cp\u003eIn present study, the correlation analysis indicated that the increase of DPPH\u0026middot; scavenging rate showed a significant positive correlation with the level of demethyltexasin (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9816, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The demethyltexasin was a trihydroxyisoflavone, the -OH group on site 6 (A-ring) in demethyltexasin acted as a primary radical scavenging site [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, demethyltexasin had a significant hyaluronic acid inhibition rate, due to the hydroxyl group on site 6 and 7 of the isoflavone skeleton [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Besides, our result indicated that the increase of \u0026middot;OH scavenging rate was positively correlated with the levels of ferulic acid (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9848, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), chlorogenic acid (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9648, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and eriodictyol (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9986, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB C D). The ferulic acid and chlorogenic acid as common phenolic acids, exhibited potent antioxidant activities that were dependent on their chemical construction, such as the ring substituents, degree of methoxylation and the number of -OH groups [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Eriodictyol was a flavonoid that belongs to a subclass of flavanones, it had the potential to enhance cell protection through increased antioxidant activity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In an in vivo study, eriodictyol showed decreased oxidative damage in human retinal pigment epithelial-19 cells by Nrf2, and the activation of its downstream shielding phase-II enzymes, namely, heme oxygenase 1 (HO-1) and NADPH quinone dehydrogenase 1 (NQO1) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003e3.8.2. Correlation analysis between cytoprotection activity and differential metabolites\u003c/h2\u003e\u003cp\u003eIn this study, the correlation analysis found that the taxifolin (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9705, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), paeonol (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9533, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and riboflavin (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9924, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eE F G) were positively correlated with cytoprotection activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The cytoprotective effect of taxifolin was investigated in previous research [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Taxifolin could suppress the activity of cathepsin D, B, PARP, caspase 3, -7, and cell cycle-related proteins and by activating the hsp27 to protect HaCaT cells against damage [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The cytoprotective effect of paeonol has not been studied well. However, paeonol could alleviate renal injury in mice by regulating Nrf2 (NF-E2 p45-related factor 2) and NF-κB pathways [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], due to transcription factor Nrf2 regulates the cellular redox homoeostasis and cytoprotective responses [45].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eFermentation with \u003cem\u003eL. plantarum\u003c/em\u003e Picp-2 and \u003cem\u003eS. cerevisiae\u003c/em\u003e SY was an effective approach to improve the antioxidant and cytoprotection activities of \u003cem\u003eL. japonicae\u003c/em\u003e flos. Metabolomic and correlation analysis have indicated that the enhancement of antioxidant activities was attributed to demethyltexasin, ferulic acid, chlorogenic acid, eriodictyol. Besides, the increases of cytoprotection activity was positively correlated with taxifolin, paeonol and riboflavin. Meanwhile, this study demonstrated that \u003cem\u003eS. cerevisiae\u003c/em\u003e facilitated the growth of \u003cem\u003eL. plantarum\u003c/em\u003e during co-fermentation,it could enhance the metabolic activity of \u003cem\u003eL. plantarum\u003c/em\u003e, and promoting their fermentative action on \u003cem\u003eL. japonicae\u003c/em\u003e flos, improving the efficiency of fermentation. The untargeted metabolomics indicated that the \u003cem\u003eL. plantarum\u003c/em\u003e could increase the levels of steroidal saponins, amino acids, and alkaloids, while it could decrease the levels of quinic acid and terpene glycosides. The \u003cem\u003eS. cerevisiae\u003c/em\u003e could increase level of triterpenoid saponins. The combination of \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e exhibited a similar metabolic change to those of the individual strain fermentation groups, including the increase in the levels of benzoic acids, coumaric acids, cinnamic acids, flavonoids and amino acids, however, the magnitude of these changes was relatively small. Additionally, metabolomic analysis results indicate that during the fermentation process, phenolic glycosides are hydrolyzed and transformed into phenolic compounds with higher activity.\u003c/p\u003e\u003cp\u003eThis study represents the first attempt to uncover the biotransformation pathways during the fermentation process of \u003cem\u003eL. japonicae\u003c/em\u003e flos through metabolomic profiling, and identifying two potential conversion pathways. This investigation provides a molecular-level analysis of the metabolic changes in \u003cem\u003eL. japonicae\u003c/em\u003e flos during fermentation and elucidates the reasons behind the enhancement of efficacy, provides a reference for obtaining \u003cem\u003eL. japonicae\u003c/em\u003e flos and other plant-based co-fermented products with enhanced antioxidant and cell protective activities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConsent for publication\u003c/h2\u003e\u003cp\u003eThe 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, and agree to publish this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the the Young Scientists of Hunan Province (2022RC1151), China Agriculture Research System for Bast and Leaf Fiber Crops (CARS-16-E25), the Training Program for Excellent Young Innovators of Changsha (KQ2106095) and Agricultural Science and Technology Innovation Program of China (ASTIP-IBFC-06).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYiwen Wang, Dengfan Lin: Writing-Original Draft, Conceptualization, Investigation, Formal analysis; Zuohua Zhu, Wenbing Gong: Software, Validation; Yingjun Zhou, Yan Li, Zhenxiu Hu, Shaowei Yan, Chang Gao, Qiming Wang: Resources, Supervision; Yuande Peng, Chunliang Xie: Methodology, Project administration, Writing-Review \u0026amp; Editing\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi Y, Li W, Fu C et al (2020) \u003cem\u003eLonicerae japonicae\u003c/em\u003e flos and \u003cem\u003eLonicerae flos\u003c/em\u003e: a systematic review of ethnopharmacology, phytochemistry and pharmacology [J]. 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Biochem J 457:415\u0026ndash;424\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biotransformation, Lactiplantibacillus plantarum, Lonicerae japonicae flos, Metabolomics, Saccharomyces cerevisiae","lastPublishedDoi":"10.21203/rs.3.rs-7563014/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7563014/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eABSTRAT\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eLonicera japonica\u003c/em\u003e is a medicinal food rich in polyphenols. However, most of the polyphenols are in the bound state, thus resulting in low extraction efficiency. In this study, the antioxidant and cytoprotective activities of \u003cem\u003eL. japonica\u003c/em\u003e were improved after fermentation, through metabolomics, it was found that the level of some polyphenols increased, which revealed the reason for the enhanced antioxidant and cytoprotective activities of \u003cem\u003eL. japonicum\u003c/em\u003e after fermentation. Results showed that the \u0026middot;OH scavenging rate of Picp-2 and co-fermentation groups increased of 23% and 13%, the DPPH\u0026middot; scavenging rate of SY group increased of 10%, and the cytoprotection activity of co-fermentation group increased of 32%. Besides, metabolomics based on LC/MS identified a total of 576, 358, 651 differential metabolites in Picp-2, SY and co-fermentation groups respectively. The levels of benzoic acids, coumaric acids, cinnamic acids, fatty acids and flavonoids in Picp-2 and SY groups significantly increased. The Picp-2 group separately detected the increase of amino acids, quinic acids, terpene glycosides, and catechin. The changes of metabolites level in co-fermentation group were similar to the single strain groups, while the change fold was smaller. Moreover, the correlation analysis revealed a significantly positive correlation between the contend of demethyltexasin, ferulic acid, chlorogenic acid, and eriodictyol with antioxidant activity. Additionally, the amount of taxifolin, paeonol, and riboflavin were significantly positively correlated with cytoprotection activity. This study is the first to find that co-fermentation with \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e could improve the cytoprotective activity of \u003cem\u003eL. lonicerae\u003c/em\u003e, and revealed the potential biotransformation pathway of active substances through metabolomics.\u003c/p\u003e","manuscriptTitle":"Synergistic Enhancement of Antioxidant and Cytoprotective Activities in Lonicerae Japonicae Flos via Co-fermentation: Metabolomics Unraveling Key Biotransformation Pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 16:58:08","doi":"10.21203/rs.3.rs-7563014/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-09T14:56:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T14:28:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T16:15:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T08:24:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T09:19:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84703915159455089310613224645157963657","date":"2025-09-25T14:05:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98309281413872930688569669690177397694","date":"2025-09-22T13:23:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192441421023995332200222997138185875814","date":"2025-09-20T00:29:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92652771764496039203358180629148121411","date":"2025-09-19T13:00:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303242855574504137275968176867005051909","date":"2025-09-19T08:35:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-17T21:17:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-15T13:27:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-15T13:27:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Food Research and Technology","date":"2025-09-08T10:14:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bdaf1277-16fe-4a34-9f62-1064a9e99ec0","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-15T23:08:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 16:58:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7563014","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7563014","identity":"rs-7563014","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}