Development of a (Poly)phenol Metabolic Signature for Assessing (Poly)phenol-Rich Dietary Patterns.

The objective assessment of habitual (poly)phenol-rich diets in nutritional epidemiology studies remains challenging. This study developed and evaluated the metabolic signature of a (poly)phenol-rich dietary score (PPS) using a targeted metabolomics method comprising 105 representative (poly)phenol metabolites, analyzed in 24 h of urine samples collected from healthy volunteers. The metabolites that were significantly associated with PPS after adjusting for energy intake were selected to establish a metabolic signature using a combination of linear regression followed by ridge regression to estimate penalized weights for each metabolite. A metabolic signature comprising 51 metabolites was significantly associated with adherence to PPS in 24 h urine samples, as well as with (poly)phenol intake estimated from food frequency questionnaires and diaries. Internal and external data sets were used for validation, and plasma, spot urine, and 24 h urine samples were compared. The metabolic signature proposed here has the potential to accurately reflect adherence to (poly)phenol-rich diets, and may be used as an objective tool for the assessment of (poly)phenol intake.

2G-lactic acid from olive oil supply chain waste: olive leaves upcycling via Lactobacillus casei fermentation.

The transition towards a sustainable model, particularly the circular economy, emphasizes the importance of redefining waste as a valuable resource, paving the way for innovative upcycling strategies. The olive oil industry, with its significant output of agricultural waste, offers a promising avenue for high-value biomass conversion into useful products through microbial processes. This study focuses on exploring new, high-value applications for olive leaves waste, utilizing a biotechnological approach with Lactobacillus casei for the production of second-generation lactic acid. Contrary to initial expectations, the inherent high polyphenol content and low fermentable glucose levels in olive leaves posed challenges for fermentation. Addressing this, an enzymatic hydrolysis step, following a preliminary extraction process, was implemented to increase glucose availability. Subsequent small-scale fermentation tests were conducted with and without nutrient supplements, identifying the medium that yielded the highest lactic acid production for scale-up. The scaled-up batch fermentation process achieved an enhanced conversion rate (83.58%) and specific productivity (0.26 g/L·h). This research confirms the feasibility of repurposing olive waste leaves for the production of lactic acid, contributing to the advancement of a greener economy through the valorization of agricultural waste. KEY POINTS: • Olive leaves slurry as it did not allow L. casei to ferment. • High concentrations of polyphenols inhibit fermentation of L. casei. • Enzymatic hydrolysis combined to organosolv extraction is the best pretreatment for lactic acid production starting from leaves and olive pruning waste.

Improvement of Bioactive Polyphenol Accumulation in Callus of Salvia atropatana Bunge.

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line-HL, and root line-RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5-50.2 mg/g dry weight) and blue (51.4-53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle.

With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

Genomics and physiology of Catenibacillus, human gut bacteria capable of polyphenol C-deglycosylation and flavonoid degradation.

The genus Catenibacillus (family Lachnospiraceae, phylum Bacillota) includes only one cultivated species so far, Catenibacillus scindens, isolated from human faeces and capable of deglycosylating dietary polyphenols and degrading flavonoid aglycones. Another human intestinal Catenibacillus strain not taxonomically resolved at that time was recently genome-sequenced. We analysed the genome of this novel isolate, designated Catenibacillus decagia, and showed its ability to deglycosylate C-coupled flavone and xanthone glucosides and O-coupled flavonoid glycosides. Most of the resulting aglycones were further degraded to the corresponding phenolic acids. Including the recently sequenced genome of C. scindens and ten faecal metagenome-assembled genomes assigned to the genus Catenibacillus, we performed a comparative genome analysis and searched for genes encoding potential C-glycosidases and other polyphenol-converting enzymes. According to genome data and physiological characterization, the core metabolism of Catenibacillus strains is based on a fermentative lifestyle with butyrate production and hydrogen evolution. Both C. scindens and C. decagia encode a flavonoid O-glycosidase, a flavone reductase, a flavanone/flavanonol-cleaving reductase and a phloretin hydrolase. Several gene clusters encode enzymes similar to those of the flavonoid C-deglycosylation system of Dorea strain PUE (DgpBC), while separately located genes encode putative polyphenol-glucoside oxidases (DgpA) required for C-deglycosylation. The diversity of dgpA and dgpBC gene clusters might explain the broad C-glycoside substrate spectrum of C. scindens and C. decagia. The other Catenibacillus genomes encode only a few potential flavonoid-converting enzymes. Our results indicate that several Catenibacillus species are well-equipped to deglycosylate and degrade dietary plant polyphenols and might inhabit a corresponding, specific niche in the gut.

Time maters: Exploring the dynamics of bioactive compounds content, bioaccessibility and antioxidant activity during Lupinus angustifolius germination.

Germination is a process that enhances the content of health-promoting secondary metabolites. However, the bioaccessibility of these compounds depends on their stability and solubility throughout the gastrointestinal tract. The study aimed to explore how germination time influences the content and bioaccessibility of γ-aminobutyric acid and polyphenols and antioxidant capacity of lupin (Lupinus angustifolius L.) sprouts during simulated gastrointestinal digestion. Gamma-aminobutyric acid showed a decrease following gastrointestinal digestion (GID) whereas phenolic acids and flavonoids exhibited bioaccessibilities of up to 82.56 and 114.20%, respectively. Although the digestion process affected the profile of phenolic acids and flavonoids, certain isoflavonoids identified in 7-day sprouts (G7) showed resistance to GID. Germination not only favored antioxidant activity but also resulted in germinated samples exhibiting greater antioxidant properties than ungerminated counter parts after GID. Intestinal digests from G7 did not show cytotoxicity in RAW 264.7 macrophages, and notably, they showed an outstanding ability to inhibit the production of reactive oxygen species. This suggests potential benefit in mitigating oxidative stress. These findings contribute to understand the dynamic interplay between bioprocessing and digestion in modulating the bioaccessibility of bioactive compounds in lupin, thereby impacting health.

Metabolomic insights into the profile, bioaccessibility, and transepithelial transport of polyphenols from germinated quinoa during in vitro gastrointestinal digestion/Caco-2 cell transport, and their prebiotic effects during colonic fermentation.

The health-promoting activities of polyphenols and their metabolites originating from germinated quinoa (GQ) are closely related to their digestive behavior, absorption, and colonic fermentation; however, limited knowledge regarding these properties hinder further development. The aim of this study was to provide metabolomic insights into the profile, bioaccessibility, and transepithelial transport of polyphenols from germinated quinoa during in vitro gastrointestinal digestion and Caco-2 cell transport, whilst also investigating the changes in the major polyphenol metabolites and the effects of prebiotics during colonic fermentation. It was found that germination treatment increased the polyphenol content of quinoa by 21.91%. Compared with RQ group, 23 phenolic differential metabolites were upregulated and 47 phenolic differential metabolites were downregulated in GQ group. Compared with RQ group after simulated digestion, 7 kinds of phenolic differential metabolites were upregulated and 17 kinds of phenolic differential metabolites were downregulated in GQ group. Compared with RQ group after cell transport, 7 kinds of phenolic differential metabolites were upregulated and 9 kinds of phenolic differential metabolites were downregulated in GQ group. In addition, GQ improved the bioaccessibilities and transport rates of various polyphenol metabolites. During colonic fermentation, GQ group can also increase the content of SCFAs, reduce pH value, and adjust gut microbial populations by increasing the abundance of Actinobacteria, Bacteroidetes, Verrucomicrobiota, and Spirochaeota at the phylum level, as well as Bifidobacterium, Megamonas, Bifidobacterium, Brevundimonas, and Bacteroides at the genus level. Furthermore, the GQ have significantly inhibited the activity of α-amylase and α-glucosidase. Based on these results, it was possible to elucidate the underlying mechanisms of polyphenol metabolism in GQ and highlight its beneficial effects on the gut microbiota.

Enhancing physicochemical, rheological properties, and in vitro rumen fermentation of starch with Melastoma candidum D. Don fruit extract.

The utilization of polyphenol-modified starch in ruminants has not undergone extensive exploration. This study aimed to investigate the impact of the complex formed between starch and Melastoma candidum D. Don fruit extract on physicochemical properties, phenol release kinetics in various buffers simulating the gastrointestinal tract, methane production, and post-rumen digestibility. The interaction between starch and M. candidum D. Don fruit extract significantly (p < 0.001) increased resistant starch and particle size diameter. The maximum phenolic release from complex between starch and M. candidum D. Don fruit extract, due to gastrointestinal tract-simulated buffers, ranged from 22.96 to 34.60 mg/100 mg tannic acid equivalent. However, rumen and abomasum-simulated buffers released more phenolic content, whereas the intestine-simulated buffer showed higher antioxidant activity (ferric ion-reducing antioxidant power). Furthermore, complex between starch and M. candidum D. Don fruit extract significantly decreased dry matter rumen digestibility (p < 0.001) and maximum methane gas production (p < 0.001).

In Vitro Digestion of Polyphenolic Compounds and the Antioxidant Activity of Acorn Flour and Pasta Enriched with Acorn Flour.

Acorn flour is a rich source of nutrients and is beneficial to human health due to, among other things, its low glycemic index and polyphenol content. In order to obtain more accurate data on the levels and activities of the substances tested after ingestion and digestion, it may be beneficial to use a simulated in vitro digestion method. Therefore, the objective of the present study was to elucidate the content of polyphenols, individual phenolic acids, flavonoids and antiradical properties of acorn flour and pasta enriched with acorn flour before and after simulated in vitro gastrointestinal digestion. The results indicate that the total polyphenol content (TPC), flavonoid content and radical scavenging activity exhibited an increasing trend following the initial digestion stage and a decreasing trend following the second stage. Nevertheless, the levels of phenolic acids demonstrated an increase in both digestion phases. The digestion processes of polyphenols in acorn flour differ significantly from those in pasta. In the case of pasta, total polyphenols, phenolic acids and flavonoids, as well as free radical scavenging properties, demonstrated a decreasing trend following each digestion stage.

Recent Advances in the Inhibition of Membrane Lipid Peroxidation by Food-Borne Plant Polyphenols via the Nrf2/GPx4 Pathway.

Lipid peroxidation (LP) leads to changes in the fluidity and permeability of cell membranes, affecting normal cellular function and potentially triggering apoptosis or necrosis. This process is closely correlated with the onset of many diseases. Evidence suggests that the phenolic hydroxyl groups in food-borne plant polyphenols (FPPs) make them effective antioxidants capable of preventing diseases triggered by cell membrane LP. Proper dietary intake of FPPs can attenuate cellular oxidative stress, especially damage to cell membrane phospholipids, by activating the Nrf2/GPx4 pathway. Nuclear factor E2-related factor 2 (Nrf2) is an oxidative stress antagonist. The signaling pathway regulated by Nrf2 is a defense transduction pathway of the organism against external stimuli such as reactive oxygen species and exogenous chemicals. Glutathione peroxidase 4 (GPx4), under the regulation of Nrf2, is the only enzyme that reduces cell membrane lipid peroxides with specificity, thus playing a pivotal role in regulating cellular ferroptosis and counteracting oxidative stress. This study explored the Nrf2/GPx4 pathway mechanism, antioxidant activity of FPPs, and mechanism of LP. It also highlighted the bioprotective properties of FPPs against LP and its associated mechanisms, including (i) activation of the Nrf2/GPx4 pathway, with GPx4 potentially serving as a central target protein, (ii) regulation of antioxidant enzyme activities, leading to a reduction in the production of ROS and other peroxides, and (iii) antioxidant effects on LP and downstream phospholipid structure. In conclusion, FPPs play a crucial role as natural antioxidants in preventing LP. However, further in-depth analysis of FPPs coregulation of multiple signaling pathways is required, and the combined effects of these mechanisms need further evaluation in experimental models. Human trials could provide valuable insights into new directions for research and application.

Exploring the Biogenic Transformation Mechanism of Polyphenols by Lactobacillus plantarum NCU137 Fermentation and Its Enhancement of Antioxidant Properties in Wolfberry Juice.

This study investigated the transformation of polyphenols, including free and bound polyphenols during the fermentation of wolfberry juice by Lactobacillus plantarum NCU137. Results indicated that fermentation significantly increased the free polyphenols content and released bound polyphenols, enhancing the antioxidant activity. Analysis showed that there were 19 free polyphenols, mainly scopoletin, pyrogallol, and dihydroferulic acid, and 16 bound polyphenols, especially p-coumaric acid, feruloyl hexoside, and caffeic acid. A significant correlation was observed between the generation and degradation of polyphenols, and specific bound polyphenols peaked during the 24-48 h fermentation. Furthermore, reduced surface roughness and galacturonic acid content in wolfberry residue, along with increased pectinase activity, suggested substantial pectin degradation in the cell wall, which may be associated with the release of polyphenols, due to pectin serving as carriers for bound polyphenols. The fermentation also increased polyphenol oxidase and peroxidase activity, contributing to polyphenol breakdown. These findings provide insights for improving wolfberry juice production.

Bound Polyphenols of Oat Bran Released by Gut Microbiota Mitigate High Fat Diet-Induced Oxidative Stress and Strengthen the Gut Barrier via the Colonic ROS/Akt/Nrf2 Pathway.

Whole-grain foods are rich in bound polyphenols (BPs) whose health benefits were largely underestimated compared with free polyphenols. We first found that DFBP (dietary fiber with BPs from oat bran) exhibited stronger colonic antioxidant activities than DF. 16S rRNA sequencing showed that DFBP selectively changed gut microbial composition, which reciprocally released BPs from DFBP. Released polyphenols from DFBP reduced excessive colonic ROS and exhibited colonic antioxidant activities via the ROS/Akt/Nrf2 pathway revealed by transcriptome and western blot analysis. Colonic antioxidant activities of DFBP mediated by gut microbiota were next proven by treating mice with broad-spectrum antibiotics. Next, Clostridium butyricum, as a distinguished bacterium after DFBP intervention, improved colonic antioxidant capacities synergistically with DFBP in HFD-fed mice. This was explained by the upregulated mRNA expression of esterase, and cellulase of Clostridium butyricum participated in releasing BPs. Our results would provide a solid basis for explaining the health benefits of whole grains.

Synergistic effects of superfine grinding and high hydrostatic pressure on the contents, distribution, digestive behaviors and antioxidant activities of polyphenols in barley leaves.

Barley leaves (BLs) naturally contained abundant phenolics, most of which are hardly completely released from food matrix during gastrointestinal digestion. Superfine grinding (SFG) and high hydrostatic pressure (HHP) are generally used to treat the functional plants due to their effectiveness to cell wall-breaking and improvement of nutraceutical bioavailability. Thus, this study investigated the synergistic effects of SFG and HHP (100, 300, 500 MPa/20 min) on the bioaccessbility of typical phenolics in BLs during the simulated in-vitro digestion. The results demonstrated that the highest bioaccessbility (40.98%) was found in the ultrafine sample with HHP at 500 MPa. CLSM and SEM confirmed SFG led to microstructurally rapture of BLs. Moreover, the recovery index of ABTS radical scavenging activity and FRAP of HHP-treated ultrafine and fine BLs samples maximumly increased by 53.62% and 9.61%, respectively. This study is expecting to provide the theoretical basis to improve the consumer acceptance of BLs.

Exploring the bioaccessibility of polyphenols and glucosinolates from Brassicaceae microgreens by combining metabolomics profiling and computational chemometrics.

Microgreens constitute natural-based foods with health-promoting properties mediated by the accumulation of glucosinolates (GLs) and phenolic compounds (PCs), although their bioaccessibility may limit their nutritional potential. This work subjected eight Brassicaceae microgreens to in vitro gastrointestinal digestion and large intestine fermentation before the metabolomics profiling of PCs and GLs. The application of multivariate statistics effectively discriminated among species and their interaction with in vitro digestion phases. The flavonoids associated with arugula and the aliphatic GLs related to red cabbage and cauliflower were identified as discriminant markers among microgreen species. The multi-omics integration along in vitro digestion and fermentation predicted bioaccessible markers, featuring potential candidates that may eventually be responsible for these functional foods' nutritional properties. This combined analytical and computational framework provided a promising platform to predict the nutritional metabolome-wide outcome of functional food consumption, as in the case of microgreens.

Sea Buckthorn Polyphenols Alleviate High-Fat-Diet-Induced Metabolic Disorders in Mice via Reprograming Hepatic Lipid Homeostasis Owing to Directly Targeting Fatty Acid Synthase.

Our previous studies found that Sea Buckthorn polyphenols (SBP) extract inhibits fatty acid synthase (FAS) in vitro. Thus, we continued to explore possible effects and underlying mechanisms of SBP on complicated metabolic disorders in long-term high-fat-diet (HFD)-fed mice. To reveal that, an integrated approach was developed in this study. Targeted quantitative lipidomics with a total of 904 unique lipids mapping contributes to profiling the comprehensive features of disarranged hepatic lipid homeostasis and discovering a set of newfound lipid-based biomarkers to predict the occurrence and indicate the progression of metabolic disorders beyond current indicators. On the other hand, technologies of intermolecular interactions characterization, especially surface plasmon resonance (SPR) assay, contribute to recognizing targeted bioactive constituents present in SBP. Our findings highlight hepatic lipid homeostasis maintenance and constituent-FAS enzyme interactions, to provide new insights that SBP as a functional food alleviates HFD-induced metabolic disorders in mice via reprograming hepatic lipid homeostasis caused by targeting FAS, owing to four polyphenols directly interacting with FAS and cinaroside binding to FAS with good affinity.

Dynamic changes in the non-volatile and flavour compounds in withered tea leaves of three different colour cultivars based on multi-omics.

In this study, metabolomics and proteomics were performed to investigate the fluctuations of non-volatile compounds and proteins in tea leaves from three tea cultivars with varying colours during withering. A total of 2798 compounds were detected, exhibiting considerable variations in amino acids, phenylpropanoids, and flavonoids. The ZH1 cultivar displayed increased levels of amino acids but decreased levels of polyphenols, which might be associated with the up-regulation of enzymes responsible for protein degradation and subsequent amino acid production, as well as the down-regulation of enzymes involved in phenylpropanoid and flavonoid biosynthesis. The FUD and ZH1 cultivars had elevated levels of flavanols and flavanol-O-glycosides, which were regulated by the upregulation of FLS. The ZJ and ZH1 cultivars displayed elevated levels of theaflavin and peroxidase. This work presents a novel investigation into the alterations of metabolites and proteins between tea cultivars during withering, and helps with the tea cultivar selection and manufacturing development.

Characterization of polyphenols and carbohydrates exuded by Phaeodactylum tricornutum diatom grown under Cu stress.

This study is focused on analysing polyphenols and carbohydrates released by Phaeodactylum tricornutum (P. tricornutum) diatoms cultured in natural seawater enriched with sublethal and lethal Cu doses. Cu concentrations of 0.31, 0.79 and 1.57 µM reduced cell densities by 37, 82 and 91%, respectively, compared to the control. The total sum of all identified polyphenols and total carbohydrates released by cells grown under lethal Cu levels increased up to 18.8 and 107.4 times, respectively, compared to data from a control experiment. Four different in vitro assays were used to estimate the antioxidant activities of the extracellular compounds: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition, cupric ion reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power and Cu complexing ability (CCA). The highest antioxidant activities were observed in the Cu lethal treatments, where the CCA assay exhibited a greater increase (up to 32.2 times higher than that found in the control experiment) to reduce the concentration of free Cu in the medium and its toxicity. The presence of Cu stimulated the release of polyphenols and carbohydrates to the medium as a detoxification mechanism to survive under lethal levels of Cu regulating its speciation.

How do in vitro digestion and cell metabolism affect the biological activity and phenolic profile of grape juice and wine.

Cabernet Sauvignon grape juice and wine underwent in vitro digestion, resulting in a reduction of most phenolic compounds (10%-100% decline), notably impacting anthocyanins (82%-100% decline) due to pH variations. However, specific phenolics, including p-hydroxybenzoic, protocatechuic, vanillic, p-coumaric, gallic and syringic acids, and coumarin esculetin, increased in concentration (10%-120%). Grape juice and wine samples showed comparable polyphenolic profile during all phases of digestion. Antioxidant activity persisted, and inhibition of angiotensin-I converting enzyme was improved after the digestion process, likely because of increased concentrations of listed phenolic acids and esculetin. Digested grape juice displayed comparable or superior bioactivity to red wine, indicating it as a promising source of accessible grape polyphenols for a broader audience. Nevertheless, Caco-2 cell model metabolization experiments revealed that only 3 of 42 analyzed compounds passed to the basolateral compartment, emphasizing the significant impact of digestion on polyphenol bioactivity, suggesting potential yet unmeasurable and overlooked implications for human health.

Phytochemical Profile and Bioactivity of Bound Polyphenols Released from Rosa roxburghii Fruit Pomace Dietary Fiber by Solid-State Fermentation with Aspergillus niger.

This study aimed to investigate the phytochemical profile, bioactivity, and release mechanism of bound polyphenols (BPs) released from Rosa roxburghii fruit pomace insoluble dietary fiber (RPDF) through solid-state fermentation (SSF) with Aspergillus niger. The results indicated that the amount of BPs released from RPDF through SSF was 17.22 mg GAE/g DW, which was significantly higher than that achieved through alkaline hydrolysis extraction (5.33 mg GAE/g DW). The BPs released through SSF exhibited superior antioxidant and α-glucosidase inhibitory activities compared to that released through alkaline hydrolysis. Chemical composition analysis revealed that SSF released several main compounds, including ellagic acid, epigallocatechin, p-hydroxybenzoic acid, quercetin, and 3,4-dihydroxyphenylpropionic acid. Mechanism analysis indicated that the disruption of tight structure, chemical bonds, and hemicellulose was crucial for the release of BPs from RPDF. This study provides valuable information on the potential application of SSF for the efficient release of BPs from RPDF, contributing to the utilization of RPDF as a functional food ingredient.

The effect of rice protein-polyphenols covalent and non-covalent interactions on the structure, functionality and in vitro digestion properties of rice protein.

The characteristics of the crosslinking between rice protein (RP) and ferulic acid (FA), gallic acid (GA), or tannin acid (TA) by covalent binding of Laccase and non-covalent binding were evaluated. The RP-polyphenol complexes greatly improved the functionality of RP. The covalent effect with higher polyphenol binding equivalence showed higher emulsion activity than the non-covalent effect. The solubility, and antioxidant activity of covalent binding were higher than that of non-covalent binding in the RP-FA group, but there was a contrasting behavior in the RP-GA group. The RP-FA was most soluble in conjugates, while the RP-GA had the highest solubility in mixtures. It was found that the covalent complexes were more stable in the intestinal tract. The content of polyphenols in the RP-TA group was rapidly increased at the later intestinal digestion, which indicated the high polyphenol-protective effect in this group. Meanwhile, the RP-TA group showed high reducing power but low digestibility.