Spatial distribution of antioxidant activity in baguette and its modulation of proinflammatory cytokines in RAW264.7 macrophages

The baguette’s distinct crust and crumb arise from Maillard reactions during baking, which shape sensory attributes but whose biological impacts remain insufficiently characterized. This study asks how Maillard-derived products within baguette, distributed from crumb to crust, influence antioxidant capacity and immunomodulatory effects in immune cells. The authors posit that the degree of Maillard reaction varies spatially within bread and may modulate cellular oxidative stress and inflammatory responses. Given the prevalence of macrophages in the intestinal mucosa and prior links between diet, oxidative stress, and inflammatory cytokines affecting appetite and metabolism, the work aims to establish a cell-based model to elucidate interactions between baguette components and macrophage function, and to inform nutritional recommendations for baked foods.

Prior research shows Maillard reaction products (MRPs), including melanoidins, form during baking and can have antioxidant and potential anti-inflammatory effects. Bread crust melanoidins can influence gut microbial communities and may inhibit enterobacteria. Pronylated lysine in crust melanoidins can induce glutathione S-transferases, indicating antioxidant and chemopreventive activity. Conversely, MR can produce undesirable compounds like AGEs and acrylamide. Studies on food-derived nanoparticles (e.g., from bone soup and vinegar) show modulation of immune cell activity and oxidative stress in the gut. Bread-making variables affect MRP formation and antioxidant capacity, with crust generally richer in late-stage MRPs and antioxidants than crumb. However, comprehensive cell-based evaluations of baguette crust versus crumb on macrophage oxidative and inflammatory responses have been limited.

Baguette preparation: Dough formulation used 450 g high-gluten flour, 4 g dry yeast, 2.25 g bread improver, 4.5 g salt, and 342 g water. Mixed 10 min, fermented 3.5 h, baked in a steam oven at 230 °C for 30 min. Breads rested 24 h at room temperature and were stored at −20 °C. Sampling and extraction: Crust (BCst) and crumb (BCmb) were separated from both ends (2 cm from ends) and midsection (4 cm). Samples were dried, milled, sieved (0.15 mm), and stored at −20 °C. Aqueous extracts (BCstE, BCmbE) were prepared by mixing 3 g powder with 30 mL deionized water, vortexing 30 min, standing 30 min, centrifuging (5813 g, 15 min, 25 °C); supernatants collected and stored at 4 °C. Dry matter determined after drying at 105 °C for 3 h. Physicochemical analyses: Protein (BCA), carbohydrates (anthrone-sulfuric acid, glucose standard), triglycerides (GPO-PAP kit), total phenolics (Folin–Ciocalteu), turbidity (A600). Color metrics L*, a, b* measured with a Chroma Meter; yellowing index (YI) calculated as YI = 142.86 × b*/L*. Fluorescent AGEs: Bread powders were digested (Tris-HCl, proteinase E, SDS, CaCl2; 30 °C, 36 h), centrifuged, diluted, and measured by fluorescence spectroscopy at Ex/Em 370/440 nm (glycosylated collagen), 335/385 nm (pentosidine), and 330/420 nm (pyrrole/imidazole derivatives). Extraction yields calculated by comparing extract fluorescence to original matrix. Antioxidant assays: ORAC performed using fluorescein and AAPH with Trolox standards, reading Ex485/Em520 nm over 180 min; results expressed as TE µmol/g. Total antioxidant capacity measured by ABTS and FRAP kits. Spatial distribution mapping: Cross-sections were grid-sampled: middle section into 5 transverse × 3 longitudinal zones (sites 1–15), and each end section into 4 transverse × 3 longitudinal zones (sites A–L). ORAC and fluorescent AGEs were measured per site to generate heat maps. Cell culture: RAW 264.7 macrophages cultured in DMEM + 10% FBS + 1% pen/strep (5% CO2, 37 °C), passages 10–20. Cytotoxicity: MTT assay after 24 h exposure to serial dilutions of BCstE/BCmbE; absorbance at 570 nm. LDH cytotoxicity assay measured extracellular LDH; LDH enzymatic activity assayed in supernatants after 24 h exposure. Cellular antioxidant activity (CAA): Cells co-incubated with DCFH-DA (50 µM) and sample or quercetin standards for 1 h, then challenged with AAPH (600 µM). Fluorescence read every 5 min for 1 h (Ex485/Em538). CAA units calculated as (1 − AUCsample/AUCcontrol) × 100; expressed as µmol/g quercetin equivalents from calibration. Membrane potential and mitochondrial ROS: Cells stained with Mito-SOX Red (3 µM, 10 min) and DiBAC4(3) (5 µM, 15 min). Samples added at various dilutions; AAPH (6.4 mM) used to induce oxidative stress. Fluorescence recorded every 10 min for 120 min (DiBAC4(3) Ex493/Em516; Mito-SOX Ex510/Em580); fluorescence microscopy captured representative images. Inflammatory cytokines: Cells incubated 24 h with BCstE or BCmbE (1:5, 1:40 dilutions). Supernatants analyzed by ELISA for TNF-α, IL-1β, IL-6. For LPS challenge, cells pretreated with LPS (40 µg/mL, 1 h) then co-incubated 24 h with extracts; IL-1β and IL-6 quantified. Statistics: Experiments in triplicate; data as mean ± SD. One-way ANOVA with Tukey’s post-hoc; significance at p < 0.05.

- Chemical composition (aqueous extracts): BCstE vs BCmbE (mean ± SD). Dry matter: 7.46 ± 0.07 vs 8.57 ± 0.05 mg/mL; Protein: 0.77 ± 0.03 vs 0.43 ± 0.02 mg/mL; Carbohydrate: 5.36 ± 0.12 vs 5.95 ± 0.06 mg/mL; Triglycerides: 3.67 ± 0.15 vs 4.00 ± 0.02 mmol/L; Total phenolics: 38.47 ± 0.79 vs 25.29 ± 0.40 µg/mL; Turbidity: 74.00 ± 1.00 vs 143.67 ± 2.08 mAbs (all p < 0.05). - Browning and color: Crust exhibited greater browning than crumb. Extract YI: BCstE 17.81 vs BCmbE 10.61 (p < 0.05), consistent with more advanced Maillard reaction in crust. - Fluorescent AGEs: Higher in crust than crumb in both matrix and extracts. In matrix (AU): BCst vs BCmb: pentosidine 63,770 ± 585 vs 9,911 ± 80; glycosylated collagen 54,670 ± 272 vs 13,380 ± 69; pyrrole/imidazole derivatives 76,880 ± 437 vs 17,170 ± 100. In extracts: BCstE vs BCmbE: pentosidine 10,374 ± 112 vs 1,788 ± 7; glycosylated collagen 14,524 ± 109 vs 2,802 ± 19; pyrrole/imidazole derivatives 19,212 ± 130 vs 4,232 ± 33 (all p < 0.05). - AGE extraction yields (%): Glycosylated collagen 16.27 ± 0.04 (BCstE) vs 18.04 ± 0.08 (BCmbE); Pentosidine 26.57 ± 0.07 vs 20.94 ± 0.06; Pyrrole/imidazole derivatives 24.99 ± 0.05 vs 24.65 ± 0.07. - Antioxidant capacity: Crust extract showed significantly higher ORAC, ABTS, and FRAP values than crumb extract. Strong positive correlations among ORAC, ABTS, FRAP, total phenolics, YI, and fluorescent AGEs (Table 5; many r ≥ 0.9, p < 0.01). - Spatial distribution: Antioxidant capacity and fluorescent AGEs increased from crumb to crust. Middle sections (sites 12–14) exhibited higher values than end sections (J, K), likely due to non-uniform oven heat and steam distribution. - Cytocompatibility: MTT cell viability remained >80% and <110% across tested dilutions for both extracts; LDH release decreased with higher extract concentration, suggesting protective effects on membrane integrity. - Cellular antioxidant activity (CAA): BCstE exhibited greater intracellular antioxidant activity than BCmbE; quercetin equivalents 8.83 ± 0.52 µmol/g (BCstE) vs 2.21 ± 0.94 µmol/g (BCmbE). - Membrane potential and mitochondrial ROS under oxidative stress: AAPH caused membrane hyperpolarization and reduced Mito-SOX fluorescence (suppressed mitochondrial respiration). Both extracts dose-dependently counteracted these effects; BCstE mitigated membrane hyperpolarization by up to 91% (1:5 dilution) and fully restored mitochondrial respiration (100%), while BCmbE restored up to 79%. - Basal cytokine secretion: Neither extract induced IL-1β or IL-6; BCmbE did not induce TNF-α. BCstE induced low TNF-α (3.02 ± 1.56 pg/mL at 1:5; 3.20 ± 2.07 pg/mL at 1:40). - LPS-stimulated cytokines: LPS alone induced IL-1β 26.11 ± 13.84 pg/mL and IL-6 39.53 ± 3.55 pg/mL. Co-incubation with extracts reduced cytokines: BCstE (1:5) IL-1β 18.05 ± 2.42, IL-6 16.18 ± 2.29; BCstE (1:40) IL-1β 7.80 ± 5.72, IL-6 4.53 ± 7.86. BCmbE showed stronger inhibition: (1:5) IL-1β 2.97 ± 4.01, IL-6 ND; (1:40) IL-1β 1.75 ± 3.47, IL-6 ND.

The results support the hypothesis that spatially variable Maillard reactions within baguette enhance antioxidant capacity toward the crust and influence macrophage responses. Crust, richer in late-stage MRPs (e.g., melanoidins, pronylated lysine) and fluorescent AGEs, exhibited greater antioxidant capacity and protected RAW264.7 macrophages from AAPH-induced oxidative damage by normalizing membrane potential and restoring mitochondrial respiration. However, the higher AGE (and possibly protein/gluten) content in crust extract may engage RAGE or immune pathways, leading to modest TNF-α induction under basal conditions. In contrast, crumb extract, despite lower antioxidant metrics, more effectively suppressed LPS-induced IL-1β and IL-6, indicating distinct anti-inflammatory mechanisms or differential component profiles between crumb and crust. Correlation analyses linking antioxidant capacity with YI, phenolics, and AGEs reinforce the Maillard reaction’s contribution to bioactivity. The spatial heat maps further reveal processing-driven heterogeneity (middle vs ends), highlighting bakery process variables that can shape functional attributes. Collectively, the data illustrate that baguette’s crust and crumb confer complementary biological effects: robust antioxidant cytoprotection (crust) and stronger attenuation of stimulated proinflammatory cytokines (crumb).

This study delineates the spatial distribution of antioxidant capacity and AGEs across baguette cross-sections and demonstrates distinct bioactivities of crust and crumb aqueous extracts in macrophages. Crust shows higher Maillard-derived browning, phenolics, fluorescent AGEs, and antioxidant capacity, providing strong cellular protection against oxidative stress and mitochondrial dysfunction. Crumb exhibits superior suppression of LPS-induced IL-1β and IL-6, suggesting distinct anti-inflammatory potential. These findings provide a cell-based framework to evaluate baked foods’ immunomodulatory effects and inform nutritional recommendations balancing sensory qualities with health impacts. Future work should identify specific chemical drivers (melanoidins, pronyl-lysine, phenolics, gluten fractions), quantify non-fluorescent AGEs, assess bioaccessibility and digestion, and optimize baking conditions to maximize benefits while minimizing potential risks (e.g., excessive AGE formation).

- Aqueous extraction may underrepresent insoluble components and lipophilic antioxidants; composition values were lower than literature. - Fluorescence-based AGE measurements exclude non-fluorescent AGEs (e.g., CML, CEL) and may be affected by extraction/digestion efficiency; overall AGE extraction yields were low (~16–27%). - In vitro macrophage model (RAW264.7) limits direct extrapolation to in vivo human responses; gut digestion, microbiota metabolism, and bioavailability were not assessed. - Heat and steam distribution during baking caused spatial heterogeneity, complicating generalization across batches. - TNF-α induction by crust extract was observed but underlying contributors (AGE–RAGE signaling vs gluten proteins) were not resolved. - Exact antioxidant values from ORAC/ABTS/FRAP were comparative rather than absolute in figures, limiting quantitative cross-study comparisons.