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

Baguette, known for its crisp crust and soft crumb, is a global staple whose properties are significantly shaped by the Maillard reaction during baking. While the Maillard reaction's impact on sensory attributes (color, texture, flavor) is well-documented, its influence on the nutritional and health aspects, particularly the biological effects on cells, remains largely unexplored. This study addresses this gap by investigating the spatial distribution of antioxidant capacity within the baguette (crust vs. crumb) and its subsequent effects on inflammation, specifically focusing on the modulation of proinflammatory cytokines in RAW264.7 macrophages. Understanding these biological impacts is crucial for refining nutritional recommendations and further elucidating the complex relationship between diet, immune responses, and overall health. The research posits that the Maillard reaction plays a key role in influencing the antioxidant and anti-inflammatory properties of baguette, and that these properties are not uniformly distributed throughout the bread. This uneven distribution could impact the overall biological activity and health benefits associated with baguette consumption. The study utilizes a cell-based approach to assess the impacts of baguette extracts on macrophage activity, which are immune cells with crucial roles in inflammation and metabolic disorders within the intestinal mucosa, a site with high macrophage density and involvement in both inflammation and metabolic function.

Existing literature extensively details the Maillard reaction's role in shaping bread's sensory characteristics, such as color and texture. However, there's a significant gap in understanding its influence on the biological effects, specifically its impact on antioxidant and anti-inflammatory properties. Studies have shown that Maillard reaction products (MRPs), including melanoidins, possess various bioactivities, such as antioxidant and potential anti-inflammatory effects through inhibition of enterobacteria. Pronylated lysine, found in bread melanoidins, induces Glutathione S-transferases, a parameter for antioxidant and chemopreventive activity. Although the Maillard reaction introduces some negative aspects (AGEs, acrylamide), careful baking control can mitigate these effects. Research highlights the importance of diet in modulating immune responses and regulating appetite, suggesting a potential link between health-promoting diets and consumer preferences. The beneficial impacts of diets like porcine bone soup and polyphenol-rich aged vinegar are mentioned, highlighting their immunomodulatory effects and ability to reduce cellular oxidative stress. This research aims to expand upon this previous work to more fully understand the role of baguette components in the modulation of immune cell activity.

The study used baguettes made with high-gluten flour, yeast, and improver. After baking and a 24-hour rest, the baguettes were frozen until analysis. Baguette crust and crumb samples were collected, dried, and ground. Aqueous extracts were prepared by mixing the powder with deionized water, vortexing, and centrifuging. The chemical composition of the extracts (dry matter, protein, carbohydrate, triglyceride, total phenols, turbidity) was analyzed using various kits and spectrophotometry. Browning intensity was determined using a chroma meter and the yellowing index. Fluorescent advanced glycation end products (AGEs) were quantified by fluorescence assays. Antioxidant capacity was measured using ORAC, ABTS, and FRAP assays. Spatial distribution of antioxidant capacity and AGEs was assessed by grid sampling across baguette cross-sections. RAW264.7 macrophages were used to evaluate cytotoxicity (MTT and LDH assays), cellular antioxidant activity (CAA), cell membrane potential (DiBAC4(3)), mitochondrial superoxide (Mito-SOX Red), and inflammatory cytokine production (ELISA). Statistical analysis involved one-way ANOVA followed by Tukey's test. Specific assays employed were bicinchoninic acid (BCA) for protein, anthrone-sulfuric acid for carbohydrates, and triglyceride kits for triglycerides. Total phenolic content (TPC) utilized Folin-Ciocalteau reagent. Fluorescence assays at specific excitation/emission wavelengths were used for different AGEs. ORAC, ABTS and FRAP assays were used to determine antioxidant capacity. MTT and LDH assays were used for cytotoxicity assessment, and an ELISA kit for pro-inflammatory cytokine quantification. AAPH was used to induce oxidative stress in the cell experiments.

The baguette crust extracts (BCstE) had significantly lower dry matter but higher protein and phenolic compound concentrations compared to crumb extracts (BCmbE). BCstE showed greater browning intensity (higher yellowing index) and AGE concentrations than BCmbE. Antioxidant capacity, assessed by ORAC, ABTS, and FRAP, was significantly higher in BCstE. Both extracts exhibited cytocompatibility with macrophages and reduced cellular oxidative stress. However, BCstE was substantially more effective in mitigating peroxyl radical-induced cell membrane hyperpolarization (91% reduction) and completely neutralized the suppression of mitochondrial oxygen respiration. In contrast, BCmbE was more potent in inhibiting lipopolysaccharide (LPS)-induced IL-1β and IL-6 expression in macrophages. BCstE, at high concentrations, induced TNF-α production, while BCmbE did not. The spatial distribution analysis showed a gradual increase in antioxidant capacity and AGEs from the crumb to the crust, with lower values at the baguette ends, potentially due to uneven heat distribution during baking. Correlation analysis revealed strong positive correlations between antioxidant capacity, browning intensity, AGE content, and total phenolic content.

The findings demonstrate a clear spatial variation in the antioxidant and anti-inflammatory properties of baguette, primarily driven by the Maillard reaction, which is more pronounced in the crust. The higher antioxidant capacity of the crust is likely due to a greater accumulation of Maillard reaction products with high antioxidant power such as melanoidins. The crumb extracts' superior anti-inflammatory properties, despite lower antioxidant activity, suggest other mechanisms are involved, potentially related to specific components that can effectively modulate proinflammatory cytokine release in macrophages. The observation of BCstE-induced TNF-α production highlights a potential trade-off between the antioxidant and pro-inflammatory effects of certain Maillard reaction products. The spatial variability observed in antioxidant capacity and AGEs likely reflects differences in heat exposure across the baguette during baking. This study introduces the use of CAA assay for baguette, providing a more accurate assessment of its intracellular antioxidant activity. The observed effects may be due to a combination of factors including AGE content, protein components (possibly gluten), and the balance between antioxidant and pro-inflammatory activities.

This study reveals a spatial heterogeneity in the bioactive properties of baguette, with the crust exhibiting superior antioxidant activity and the crumb demonstrating better anti-inflammatory properties in macrophages. The Maillard reaction is a major contributor to these differences. This research provides valuable insights into the complex relationship between baguette consumption and immune responses, offering important data for the refinement and supplementation of dietary guidelines. Future research could focus on identifying the specific components responsible for the observed anti-inflammatory activity in the crumb, exploring the long-term effects of baguette consumption on the immune system, and investigating the impact of different baking processes on the distribution of bioactive compounds. Further investigation into the effects of non-fluorescent AGEs would also be valuable.

The study used only one type of baguette, limiting the generalizability of the findings to other bread types. The aqueous extraction method might not have extracted all bioactive compounds from the baguette, potentially underestimating the actual content. The use of a cell line (RAW264.7 macrophages) instead of primary cells in vitro limits the translational potential. The research focused on a specific subset of AGEs, and other types could have contributed additional effects. The study’s in vitro nature limits direct extrapolation to human in vivo effects. Further research is needed to confirm and extend these findings.