DOI: 10.6084/m9.figshare.29386646.v2
title: <b>Figure 1; 2; 3; 4; 5; 6</b><b>Table 1, </b> 2, 3<b>; </b><b>Sup Table</b> 1,2,3,4; <b>Sup Figure</b> 1,2

description: <b>Figure 1.</b> Mechanistic overview of bioactive peptides derived from <i>L. reuteri</i>-fermented and germinated brown rice. These peptides exert multifunctional health benefits, including antioxidant, anti-inflammatory, anti-obesity, and gut-modulating effects, by modulating pathways like P2X7R and TLR2/4. They enhance immune responses through ERK1/2 and Akt-p38 signaling, ultimately reducing pro-inflammatory cytokines and promoting cellular resilience.<b>Figure 2.</b> Multifunctional neuroprotective mechanisms of <i>L. reuteri</i> fermented brown rice peptides targeting key pathways in neurodegeneration. These include antioxidant defense, anti-inflammation, autophagy enhancement, neurotransmitter regulation, and gut microbiome modulation. Collectively, they mitigate amyloid plaques, tau tangles, oxidative stress, and neuro-inflammation to preserve cognitive function.<b>Figure 3.</b> Nano-incorporation of <i>L. reuteri</i> fermented bioactive peptides enhances therapeutic potential against neurodegenerative disorders by overcoming limitations such as low stability, poor bioavailability, and rapid clearance. Nanodelivery improves peptide absorption, targeted cellular uptake, and sustained release, enabling applications in neuroprotection, gut health, and smart functional foods.<b>Figure 4. </b>Advanced strategies for targeted delivery of bioactive peptides via nano-formulations. These include pH-sensitive enteric release for intestinal targeting, enzyme-responsive cleavage for microbial enzyme-triggered release, ligand-mediated mucosal targeting for receptor-specific uptake, and combined spatiotemporal control using dual-triggered nanocarriers for precision delivery along the GI tract.<b>Figure 5.</b><b> </b>Overview of brown rice-derived bioactive peptide modification and its impact on gut–brain axis regulation. Fermentation and enzymatic hydrolysis of brown rice proteins (albumin, globulin, prolamin, glutelin) yield low-molecular-weight peptides with high antioxidant activity. These peptides modulate gut microbiota, reduce inflammation, and influence mental health, contributing to either positive or poor brain outcomes depending on dietary patterns and microbial balance.<b>Figure 6</b>: Nano-enabled delivery model mechanisms of <i>Limosilactobacillus reuteri</i> fermented brown rice peptides across the gut–brain axis. The targeted release in the ileum or colon, enhanced intestinal absorption, and systemic distribution of neuroactive peptides. These bioactives modulate gut microbiota, stimulate short-chain fatty acid production, and influence neurotransmitter pathways (e.g., serotonin), contributing to immune resilience, metabolic balance, and cognitive function. The figure underscores the therapeutic potential of this nanoformulation strategy in advancing precision nutrition and functional food-based prevention of chronic diseases.<b>Table 1.</b> Strategies and Mechanisms for Enhancing the Absorption and Bioavailability of Nano-Encapsulated Bioactive Peptides.<b>Table 2.</b> Comprehensive mechanisms and health impacts of nano-delivered <i>Limosilactobacillus reuteri</i>-fermented brown rice peptides in stress relief and neuroinflammation prevention. The outlines of 20 mechanistic pathways targeted by key bioactive components, such as GABA, quercetin, and tryptophan, delivered based on advanced nanocarrier systems. It highlights how encapsulation improves peptide stability, absorption, targeted release, and systemic bioactivity, leading to neuroprotective, immunomodulatory, and mood-enhancing effects through gut-brain axis modulation.<b>Table 3</b>. Potential applications of nano-delivered <i>Limosilactobacillus reuteri</i> fermented brown rice peptides in functional food systems. The outlines diverse product categories, including beverages, snacks, supplements, and clinical formulations, where nanoencapsulated peptides can be incorporated. Each entry highlights the functional role, consumer relevance, and nano-delivery advantages such as enhanced bioavailability, stability, targeted release, and compatibility with various food matrices. These applications support the development of next-generation functional foods aligned with precision nutrition, gut-brain health, and chronic disease prevention.<b>Sup Table 1. </b>Identified Peptides from <i>Limosilactobacillus reuteri</i> Fermented Brown Rice. The lists of 70 peptides identified through LC-MS/MS after fermentation of brown rice by <i>L. reuteri</i>. The data includes each peptide’s sequence, precursor and theoretical molecular weights (MW), m/z values, and retention times, providing a comprehensive overview of peptide composition and elution characteristics (Tyagi et al. 2023; https://doi.org/10.1016/j.foodchem.2022.134747).<b>Sup Table 2. </b>Antioxidant Activity (IC₅₀ Values) of Selected Peptides. The in vitro antioxidant activity of six selected peptides and ascorbic acid (standard) measured using DPPH, ABTS, FRAP, and PSC assays. IC₅₀ values (µg/mL) reflect the concentration required to scavenge 50% of radicals in each assay, highlighting the radical scavenging potential of each compound (Tyagi et al. 2023; https://doi.org/10.1016/j.foodchem.2022.134747).<b>Sup Table 3.</b> Molecular Docking Parameters of Peptides with Keap1 Protein. The docking results of the six peptides and TX6 (reference ligand) with Keap1 protein (PDB ID: 2FLU). It includes binding energies, hydrogen bond residues, and hydrophobic interaction residues, providing insights into peptide–Keap1 interaction strength and stability relevant to antioxidant activity via the Nrf2 pathway (Tyagi et al. 2023; https://doi.org/10.1016/j.foodchem.2022.134747).<b>Sup Table 4</b>. Identification of bioactive peptides in differently processed brown rice samples, including raw, germinated (Germ), fermented with <i>L. reuteri</i> AKT1 (Ferm), and germinated + fermented (G+F). Peptides were characterized based on retention time, peak area, ionization mode, precursor mass, molecular formula, and putative sequence. Each peptide was associated with known physiological roles, including antioxidant, stress-reducing, sleep-inducing, and neuroprotective activities. The presence and abundance of specific peptides varied across treatments, highlighting the impact of bioprocessing on bioactive peptide formation. ND: Not Detected.Sup Figure 1. Comparative amino acid profiling in raw, germinated, fermented (L. reuteri AKT1), and germinated + fermented (G+F, L. reuteri AKT1) brown rice samples.(A) Heat map representation of amino acid composition across treatment groups, with color intensity (shades of blue) indicating relative abundance.(B) Quantification of γ-aminobutyric acid (GABA) levels among raw, germinated (Germ), fermented for 48 hours (Ferm), and combined germination + fermentation (G+F) treatments.(C) Principal component analysis (PCA) biplots illustrating sample clustering and discrimination based on amino acid profiles. PCA plots compare (left) PC1 vs. PC2 and (right) PC1 vs. PC3.Germ: germinated brown rice; Ferm: L. reuteri AKT1-fermented brown rice; G+F: germinated + fermented brown rice.Data are presented as mean ± SD from triplicate experiments using a sample concentration of 1 mg/mL. Superscripts with different letters indicate statistically significant differences (p &lt; 0.05) determined by ANOVA with post-hoc analysis using SPSS and GraphPad Prism 8.0. (Tyagi et al. 2021; https://doi.org/10.3390/ antiox10040626).<b>Sup Figure 2</b> <b>(A–G)</b>. Molecular docking analysis of identified antioxidant peptides and their interactions with the Keap1 protein (PDB ID: 2FLU). The binding modes reveal hydrogen bonds and hydrophobic interactions within the Kelch domain of Keap1. Panel <b>A</b> shows the interaction of the reference ligand TX6 (PubChem CID: 121488089) with Keap1. Panels B through G display the binding conformations of six bioactive peptides: (<b>B</b>) AVPYPQ (P1), (<b>C</b>) ILTAV (P2), (<b>D</b>) LGDVIGVP (P3), (<b>E</b>) NPIFDYVLLP (P4), (<b>F</b>) VAPFPEV (P5), and (<b>G</b>) VLPVPK (P6). TX6 binds at the canonical Keap1 site (A), while all peptides demonstrate stable attachment to Keap1 at overlapping or adjacent binding pockets (B–G), indicating potential competitive inhibition. Interaction diagrams highlight specific hydrogen bonds (green dashed lines) and hydrophobic contacts (red arcs), emphasizing key residues involved in ligand recognition. (Tyagi et al. 2023; https://doi.org/10.1016/j.foodchem.2022.134747).