Galactomannan-rich ingredients from Caesalpinia pulcherrima seeds reorganize wheat bread microstructure and enhance technological and nutritional quality

Galactomannan-rich ingredients from Caesalpinia pulcherrima seeds reorganize wheat bread microstructure and enhance technological and nutritional quality | 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 Galactomannan-rich ingredients from Caesalpinia pulcherrima seeds reorganize wheat bread microstructure and enhance technological and nutritional quality Poliana Brito de Sousa, Neilane Gomes da Rocha, Camila de Carvalho Chaves, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8977729/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Galactomannan-rich ingredients from tropical legumes are promising biomaterials to design bakery products with improved structure and nutritional quality. This study evaluated the structure–function relationships of Caesalpinia pulcherrima seed flour (FSF) and seed gel (GSF) in wheat bread. A central composite design assessed the effects of FSF (3.96–11.04%) and GSF (0.17–5.82%) on technological properties, proximate composition and microstructure. FSF and GSF increased loaf specific volume (from 2.16 to 3.87 mL g⁻¹), alveoli number and circularity, while maintaining expansion index values comparable to the control, indicating improved gas retention without impairing dough expansion. FSF-enriched breads showed higher protein (up to 17.28%), lipid (up to 10.11%), ash and fiber contents than the control, confirming FSF as the main contributor to nutritional enrichment, whereas GSF mainly affected hydration and crumb structure. pH, moisture and water activity remained within typical ranges for pan bread and compatible with standard yeast fermentation. SEM and CLSM images revealed a more continuous and cohesive gluten–starch matrix with thinner cell walls and more uniform gas cells in enriched crumbs, aligning with the improved technological performance. Overall, FSF and GSF act as functional biomaterials that valorize an underutilized tropical legume and support the development of nutritionally enhanced, structurally tailored wheat breads. functional bakery products galactomannans legume-based ingredients tropical biodiversity valorization Figures Figure 1 Figure 2 Figure 3 1. Introduction Bread is one of the most consumed staple foods worldwide, typically eaten one to three times a day depending on cultural habits and cereal cost. Its production follows standardized steps, mixing, gluten development, fermentation, baking, and packaging. Although designed to meet basic energy needs, consumer demand increasingly favors foods that support health and prevent diet-related diseases. Conventional wheat bread, however, is largely composed of refined carbohydrates and contains limited protein, fiber, minerals and vitamins [ 1 , 2 ]. A common strategy to improve nutritional quality is the partial replacement of wheat flour with plant-based ingredients such as whole grains, legume flours, by-products or natural hydrocolloids. Yet, due to their gluten-free and fiber-rich nature, these ingredients often impair dough rheology, gas retention and loaf volume when used at high levels [ 3 ]. Legume-derived materials are particularly attractive because they combine proteins, lipids, minerals and dietary fiber with polysaccharides capable of modifying the dough matrix. Among tropical legumes, Caesalpinia pulcherrima , known as flamboyant-mirim, dwarf poinciana or Barbados pride, is widely distributed in tropical regions such as Brazil, India, Malaysia and Sri Lanka and is well adapted to the Brazilian Cerrado. Various plant parts exhibit biological activities [ 4 ]. Its seeds are rich in galactomannans, soluble dietary fiber with thickening, stabilizing and film-forming properties [ 5 , 6 ]. These polysaccharides are applied in dairy desserts, edible films and fiber-enriched foods and strongly influence water binding, viscosity and phase behavior. In bread, galactomannans can interact with gluten proteins and starch granules, modulating gas cell stability, crumb structure and staling. Additionally, C. pulcherrima seed flour has been explored for biofuel and animal feed due to its protein- and lipid-rich matrix, suggesting nutritional density coupled with functional macromolecules. Despite these attributes, no studies have evaluated C. pulcherrima seed derivatives in breadmaking. This knowledge gap represents an opportunity to (i) valorize an underutilized tropical legume; (ii) investigate how its galactomannan-rich biomaterials interact with the gluten–starch network; and (iii) establish structure–function relationships relevant to enriched bakery products. Therefore, galactomannan-rich ingredients from underutilized tropical legumes such as Caesalpinia pulcherrima represent an attractive strategy to design bakery products with improved structure and nutritional quality. However, the combined effects of seed flour and gel forms on dough behavior, bread technological performance and microstructure remain unexplored. In this context, the present study aimed to elucidate the structure–function relationships of C. pulcherrima seed flour (FSF) and gel (GSF) in wheat bread, using response surface methodology to optimize their levels and advanced imaging techniques (SEM and CLSM) to link microstructural organization with technological and nutritional attributes. 2. Materials and Methods 2.1. Raw material collection Pods of Caesalpinia pulcherrima containing mature seeds were collected at the Federal University of Ceará, Campus Pici, Fortaleza, Brazil (3°44′38.6″S; 38°34′47.3″W), under SisGen authorization ABF331B. Pods were selected based on dry texture, natural dehiscence, and brown coloration. A voucher specimen was deposited in the Prisco Bezerra Herbarium (EAC 67246). 2.2. Preparation of C. pulcherrima seed flour (FSF) Pods were manually opened, and seeds were selected for integrity and absence of defects. Seeds were ground in an industrial blender (Li1.5, Skymsen, Brazil) for 5 min in 1-min intervals and then milled in a knife mill (Luca-226/5, Lucadema, Brazil) for 5 min under the same conditions. The powder was sieved through a 0.250 mm mesh using an electromagnetic shaker (Rotachoc Chopin®, France) for 10 min. The resulting seed flour (FSF; 0.250 mm) was vacuum-packed, stored at room temperature. 2.3. Preparation of C. pulcherrima seed gel (GSF) For the preparation of GSF, 20 g of seeds were mixed with 200 mL of distilled water and heated on a magnetic hot plate (Q310-22B, Quimis, Brazil) at 70°C for 5 h. After cooling to room temperature, the cooked mixture was homogenized with a vertical mixer (Black & Decker, Brazil) and filtered through a fine mesh sieve to obtain the gel. The GSF was stored in polyethylene containers at 4°C (Electrolux, Brazil). The gel yield was approximately 69.7%. 2.4. Bread formulation and experimental design The control bread formulation, without FSF or GSF, followed Guimarães et al. [ 7 ] and consisted of wheat flour (100%), water (58%), hydrogenated vegetable fat (10%), refined sugar (5%), instant dry yeast (3.3%) and salt (2%). Ingredients were purchased from a local market in Teresina, Piauí, Brazil. The experimental design consisted of eleven runs combining different concentrations of FSF and GSF according to the Central Composite Rotational Design. Run 1 was formulated with low levels of both factors (X₁ = −1; 5.0% FSF and X₂ = −1; 1.0% GSF), while Run 2 maintained the same FSF level (5.0%) combined with the high GSF level (X₂ = +1; 5.0%). Runs 3 and 4 used the high FSF concentration (X₁ = +1; 10.0%), combined with low GSF (1.0%) in Run 3 and high GSF (5.0%) in Run 4. The axial points included Run 5 (X₁ = −1.41; 3.96% FSF and X₂ = 0; 3.0% GSF) and Run 6 (X₁ = +1.41; 11.04% FSF and X₂ = 0; 3.0% GSF). Runs 7 and 8 were formulated with the central FSF level (X₁ = 0; 7.5%) combined with the lowest axial GSF level (X₂ = −1.41; 0.17%) in Run 7 and the highest axial GSF level (X₂ = +1.41; 5.82%) in Run 8. Finally, Runs 9, 10 and 11 corresponded to the central point of the design, containing 7.5% FSF and 3.0% GSF in all replicates (X₁ = 0; X₂ = 0). 2.5. Bread-making process Dough ingredients were mixed in a planetary mixer (Arno, Brazil) for 7 min, divided into 250 g portions and manually shaped. Loaves were placed in single-sheet pans and proofed at 32°C and 75% RH for 90 min in a fermentation chamber (Frilux, Brazil). Baking was performed in an electric oven (Venâncio, Brazil) at 220°C for 20 min, followed by cooling at room temperature (25 ± 2°C). Bread quality analyses were conducted 2 h after baking [ 8 ]. 2.6. Expansion index (EI) The EI was determined according to Zambelli et al. [ 8 ]. Cylindrical dough portions (~ 10 g) had their diameter and height measured with a calibrated caliper before fermentation and after baking. EI was calculated using Eq. ( 1 ): $$EI=\frac{({D}_{P}/{H}_{P})}{({D}_{M}/{H}_{M})}$$ 1 where: Dₚ – diameter of the bread after baking; Hₚ – height of the bread after baking; Dₘ – diameter of the molded dough before fermentation; Hₘ – height of the molded dough before fermentation. 2.7. Specific volume of bread Bread volume was measured by the millet seed displacement method (AACC Method 72 − 10) [ 9 ] in triplicate. Specific volume (mL.g⁻¹) was calculated as the ratio between bread volume and weight. 2.8. Crumb structure image analysis The crumb structure was analyzed by digital image processing. Central bread slices were scanned at 550 dpi using an HP ScanJet 2400, and images (900 × 900 pixels) were processed in ImageJ® 1.47v. Images were converted to 8-bit grayscale, thresholder using Otsu’s method, and analyzed for alveoli number and circularity following [ 10 ]. 2.9. Physicochemical and proximate analyses Physicochemical analyses (pH, titratable acidity, protein, moisture, ash, and crude fiber) were performed for FSF, GSF, and bread samples according to Adolfo Lutz Institute [ 11 ] and AOAC [ 12 ]. Water activity (aw) was measured using a LabSwift-aw analyzer (Novasina, Switzerland), and carbohydrate content was calculated by difference. All determinations were conducted in triplicate. 2.10. Scanning Electron Microscopy (SEM) The microstructure of FSF, GSF, and bread crumb samples was examined by SEM. Samples were fixed on aluminum stubs with carbon tape and sputter-coated with gold (~ 20 nm) using a Bal-Tec SCD 050 coater. Micrographs were obtained in a JEOL JSM-6390LV microscope operated at 15 kV, with magnifications between 500× and 2000× to visualize surface morphology, starch granules, and the protein–starch network. 2.11. Confocal Laser Scanning Microscopy (CLSM) CLSM was used to visualize the crumb microstructure and the distribution of starch and protein. Samples were mounted on glass slides and analyzed using a Zeiss LSM 710 microscope. Rhodamine B (543/560–620 nm) and FITC (488/500–540 nm) were used to label protein and starch, respectively. Images were acquired using ZEN software and processed to assess the three-dimensional organization and interactions within the protein–starch matrix. 2.12. Statistical analysis Statistical differences among treatments, control bread, and raw materials were assessed by ANOVA followed by Tukey’s test at 5% significance using Statistica® 10.0 (StatSoft Inc., USA). The effects of FSF (x₁) and GSF (x₂) on bread quality variables were modeled using second-order polynomial regression, as shown in Eq. (2). Y = β 0 + β 1 x 1 + β 2 x 2 + β 11 x 1 2 + β 22 x 2 2 + β 12 x 1 x 2 (2) where Y is the dependent variable; β₀ is the intercept; β₁ and β₂ are the linear coefficients; β₁₁ and β₂₂ are the quadratic coefficients; and β₁₂ represents the interaction coefficient between the independent variables. Model significance was assessed using the F-test, lack-of-fit, and determination coefficients (R²). Validated models were then used to generate response surface and contour plots, illustrating how FSF and GSF levels influenced the technological and nutritional attributes of the breads. This article does not contain any studies with human participants or animals performed by any of the authors. 3. Results and discussion The experimental values obtained for alveoli circularity and count, specific volume and expansion index are shown in Table 1 , together with the corresponding measurements of the control bread for comparison. Table 1 – Mean values and Tukey’s test results for technological properties of breads supplemented with FSF and GSF. Runs Alveoli circularity Alveoli number (cells) EI Specific Volume (mL.g − 1 ) 1 0.928 ± 0.02 ab 1034 ± 58.00 c 1.26 ± 0.01 a 3.27 ± 0.13 ab 2 0.900 ± 0.04 b 1120 ± 45.00 b 1.32 ± 0.11 a 3.51 ± 0.23 ab 3 0.903 ± 0.03 b 1199 ± 38.00 b 1.32 ± 0.05 a 3.55 ± 0.12 ab 4 0.939 ± 0.02 ab 1244 ± 29.00 ab 1.35 ± 0.05 a 3.59 ± 0.25 ab 5 0.832 ± 0.04 d 912 ± 31.00 c 1.28 ± 0.01 a 3.00 ± 0.13 b 6 0.952 ± 0.01 a 1383 ± 19.00 a 1.30 ± 0.04 a 3.87 ± 0.55 a 7 0.908 ± 0.03 b 1093 ± 2.00 b 1.24 ± 0.04 a 3.44 ± 0.15 ab 8 0.898 ± 0.03 c 1012 ± 32.00 bc 1.25 ± 0.05 a 3.37 ± 0.35 ab 9 0.910 ± 0.02 b 1183 ± 14.00 b 1.31 ± 0.02 a 3.60 ± 0.24 ab 10 0.905 ± 0.03 b 1179 ± 18.00 b 1.32 ± 0.03 a 3.16 ± 0.08 ab 11 0.908 ± 0.03 b 1190 ± 21.00 b 1.29 ± 0.03 a 3.53 ± 0.01 ab Control 0.639 ± 0.03 e 728 ± 30.00 d 1.31 ± 0.04 a 2.16 ± 0.05 c Different lowercase letters in the same column indicate significant differences according to Tukey’s test (p < 0.05). The incorporation of FSF and GSF increased the specific volume of all enriched breads compared with the control, indicating improved gas retention during proofing and baking. The highest specific volume (3.87 mL g⁻¹) was observed in run 6 (11.04% FSF + 3% GSF), whereas the control showed the lowest value (2.16 mL g⁻¹). Crumb image analysis further supported these macroscopic differences: breads containing FSF and GSF had higher alveoli numbers (1034–1383) and circularity (0.832–0.952) than the control (728; 0.639), resulting in a more homogeneous crumb with well-distributed and rounded gas cells [ 13 ]. Despite these structural changes, the expansion index (EI) remained statistically unchanged (1.24–1.35 cm; p > 0.05; Table 1 ), suggesting that the partial replacement of wheat flour with FSF and GSF preserved dough expansion capacity while allowing higher loaf volume and a finer crumb structure. The observed increases in loaf volume and crumb aeration are mainly related to the galactomannans naturally present in the seeds. These soluble fibers and hydrocolloids exhibit high water-holding capacity, increase dough viscosity and stabilize gas cells during fermentation. In gluten-containing systems, galactomannans interact with gluten proteins and starch granules, reinforcing the dough matrix and promoting higher loaf volume [ 5 , 14 , 15 ]. In addition to the polysaccharide effect, the slightly higher lipid contents measured in FSF- and GSF-enriched breads (8.78–10.11%) compared with the control (8.28%) may also have contributed to this behavior. Lipids can enhance dough aeration and stabilize gas bubbles by forming surface-active complexes with proteins and starch, strengthening gas-cell walls during proofing and baking. Comparable improvements in loaf volume and crumb uniformity have been reported in breads enriched with plant-based flours or hydrocolloids, which modify dough rheology and gas retention [ 8 , 16 – 18 ] In line with these studies, the present results reinforce that galactomannan-rich legume ingredients behave as functional biomaterials capable of strengthening the gluten–starch network and improving bread structure. From a biomaterials standpoint, FSF and GSF therefore act as structuring agents that generate a more cohesive and mechanically stable wheat-based crumb, explaining the improved technological performance observed. Table 2 – pH, titratable acidity, moisture, and water activity of breads with FSF and GSF, control, and raw materials. Runs pH Titratable acidity (%) Moisture (%) Water Activity 1 5.40 ± 0.01 de 0.21 ± 0.00 b 37.00 ± 0.03 b 0.952 ± 0.00 b 2 5.41 ± 0.01 cde 0.16 ± 0.00 g 35.03 ± 0.27 c 0.955 ± 0.00 b 3 5.43 ± 0.01 cde 0.20 ± 0.01 bc 32.78 ± 0.25 gh 0.955 ± 0.00 b 4 5.51 ± 0.01 b 0.20 ± 0.01 bcd 32.33 ± 0.04 h 0.956 ± 0.00 b 5 5.48 ± 0.01 bcd 0.17 ± 0.00 fg 32.88 ± 0.14 g 0.952 ± 0.00 b 6 5.52 ± 0.04 b 0.18 ± 0.00 cde 34.11 ± 0.01 ef 0.954 ± 0.00 b 7 5.55 ± 0.02 b 0.19 ± 0.00 cde 32.52 ± 0.11 gh 0.943 ± 0.00 c 8 5.48 ± 0.02 bc 0.19 ± 0.02 cde 33.79 ± 0.09 f 0.951 ± 0.00 bc 9 5.55 ± 0.02 b 0.17 ± 0.00 efg 34.82 ± 0.08 cd 0.950 ± 0.00 bc 10 5.55 ± 0.03 b 0.18 ± 0.00 cdef 34.54 ± 0.07 de 0.950 ± 0.00 bc 11 5.54 ± 0.01 b 0.18 ± 0.00 def 34.86 ± 0.38 cd 0.948 ± 0.00 bc Control 5.38 ± 0.03 e 0.30 ± 0.00 a 37.09 ± 0.10 b 0.951 ± 0.00 bc FSF 6.29 ± 0.07 a 0.12 ± 0.00 h 5.92 ± 0.12 i 0.496 ± 0.00 d GSF 6.25 ± 0.02 a 0.12 ± 0.00 h 95.24 ± 0.06 a 0.986 ± 0.00 a Different lowercase letters in the same column indicate significant differences according to Tukey’s test ( p < 0.05). The experimental results for pH, titratable acidity, moisture and water activity are presented in Table 2 for the control bread, FSF, GSF and enriched breads. FSF and GSF showed slightly acidic pH values (6.29 and 6.25, respectively), which are typical of bakery ingredients and within the range reported for C. pulcherrima seed mucilage (pH 5.7) [ 19 ]. Although near-neutral pH foods may favor spoilage microorganism growth [ 20 ], the pH values observed for FSF and GSF remain compatible with safe incorporation into bread formulations. The pH of breads containing FSF and GSF ranged from 5.40 to 5.55, while the control presented a slightly lower value (5.38). Formulations 1–3 (5.40–5.43) were similar to the control, whereas runs 4–11 exhibited higher pH values. This range is technologically adequate, since yeast fermentation is optimal between pH 4.5 and 6.5 and conventional breads typically show pH 4.7–5.4 due to organic acid formation during fermentation [ 1 ]. Previous studies have also reported only minor pH changes when plant-based ingredients are incorporated into wheat doughs [ 3 , 21 ], indicating that FSF and GSF slightly increased pH without impairing fermentation. Titratable acidity, which is related to freshness and shelf stability [ 15 ], was lowest in FSF and GSF (0.12%) and ranged from 0.16% to 0.30% in breads. Among the breads, formulation 2 exhibited the lowest acidity, whereas the control showed the highest value, in agreement with reports of reduced acidity when plant ingredients partially replace wheat flour [ 21 ]. The lower acidity of enriched breads, combined with essentially unchanged water activity values (Table 2 ), suggests that the observed differences were driven by the intrinsic composition of FSF and GSF rather than by deviations in fermentation conditions, supporting process reproducibility and compatibility with standard breadmaking. Moisture values reflected the contrasting nature of the raw materials. FSF showed low moisture (5.92%), whereas GSF presented very high moisture (95.24%), in agreement with previous reports for C. pulcherrima seeds and mucilage [ 19 , 23 – 24 ]. FSF complied with Brazilian standards for flours (maximum 15% moisture; RDC 263/2005), confirming its suitability as a dry ingredient. Bread moisture ranged from 32.33% to 37.09%, with the control displaying the highest value and formulation 4 (10% FSF and 5% GSF) the lowest. These differences can be attributed to the balance between the additional water introduced with GSF, the water-binding capacity of FSF and evaporative losses during baking. All breads met regulatory limits for pan bread moisture (≤ 38%; RDC 90/2000), indicating that FSF and GSF did not compromise product safety or legal compliance. Although several studies have reported increased moisture when plant-based ingredients are incorporated into breads [ 3 , 16 , 18 , 25 ], the present results show that, under the conditions tested, FSF and GSF levels were compatible with moisture contents typical of conventional breads. Water activity followed a complementary pattern. FSF exhibited low aw (0.496), whereas GSF showed a very high value (0.986), which explains the need for refrigeration of the gel. Bread aw ranged from 0.943 to 0.956, without significant differences (p > 0.05) relative to the control. These values confirm that all breads are high-aw products, which accounts for their naturally short shelf life and the need for appropriate packaging and storage conditions. The lack of significant changes in aw after FSF and GSF incorporation indicates that the microenvironment of available water in the crumb was not markedly altered, despite differences in formulation and moisture. Similar trends of essentially unchanged aw in enriched breads have been reported when plant-based ingredients are added to wheat-based formulations [ 22 , 26 , 27 ]. The proximate composition results (Table 3 ) highlighted marked differences in lipid content between the seed-derived ingredients. GSF had a very low lipid content (0.08%), whereas FSF exhibited a substantially higher value (9.90%), consistent with previous reports for C. pulcherrima seeds [ 22 ]. This lipid fraction is intrinsic to the seed and, when transferred to the flour, represents a natural source of fats for bakery formulations, offering the possibility of improving fatty acid profiles compared with conventional breads. Table 3 – Proximate composition and Tukey’s test for breads with FSF and GSF, control, and raw materials. Runs Lipids (%) Proteins (%) Crude Fiber (%) Ash (%) Carbohydrate (%) 1 8.78 ± 0.03 d 14.59 ± 0.23 gh 0.24 ± 0.02 de 1.62 ± 0.02 i 37.75 ± 0.45 cdef 2 9.44 ± 0.31 bc 14.81 ± 0.08 fgh 0.25 ± 0.04 de 2.03 ± 0.04 g 38.43 ± 0.52 cde 3 9.70 ± 0.06 abc 16.51 ± 0.24 bc 0.33 ± 0.01 de 2.19 ± 0.03 f 38.47 ± 0.45 cde 4 9.91 ± 0.02 ab 16.41 ± 0.22 bcd 0.35 ± 0.02 de 2.33 ± 0.02 e 38.65 ± 0.20 cd 5 8.86 ± 0.23 d 14.90 ± 0.08 fgh 0.23 ± 0.03 e 1.77 ± 0.02 h 41.34 ± 0.30 b 6 10.11 ± 0.07 a 17.28 ± 0.29 b 0.53 ± 0.04 c 2.97 ± 0.04 b 35.00 ± 0.29 h 7 9.58 ± 0.09 bc 15.95 ± 0.13 cde 0.37 ± 0.02 d 2.65 ± 0.02 d 38.93 ± 0.37 c 8 9.70 ± 0.01 abc 16.34 ± 0.15 cde 0.34 ± 0.02 de 2.79 ± 0.05 c 37.04 ± 0.16 fg 9 9.76 ± 0.10 abc 16.17 ± 0.26 cde 0.36 ± 0.02 de 2.76 ± 0.02 c 36.12 ± 0.21 gh 10 9.64 ± 0.11 abc 15.47 ± 0.42 efg 0.36 ± 0.03 de 2.72 ± 0.01 cd 37.28 ± 0.36 defg 11 9.37 ± 0.06 c 15.57 ± 0.19 def 0.37 ± 0.01 d 2.74 ± 0.02 cd 37.19 ± 0.21 efg Control 8.28 ± 0.12 e 14.20 ± 0.24 h 0.25 ± 0.00 de 1.57 ± 0.05 i 38.59 ± 0.29 cd FSF 9.90 ± 0.40 ab 27.34 ± 0.78 a 1.32 ± 0.11 a 3.85 ± 0.00 a 51.66 ± 1.27 a GSF 0.08 ± 0.01 f 2.11 ± 0.04 i 0.67 ± 0.05 b 0.15 ± 0.01 j 3.18 ± 0.06 i Different lowercase letters in the same column indicate significant differences according to Tukey’s test ( p < 0.05). In the breads, lipid contents ranged from 8.78% to 10.11%, while the control presented the lowest value (8.28%). The highest lipid level was observed in run 6 (11.04% FSF + 3% GSF), mirroring the higher FSF inclusion. Similar increases in lipid content have been reported in breads enriched with plant flours [ 17 , 27 ]. From a technological perspective, these lipids interact with gluten and starch, improving gas retention, loaf volume and crumb elasticity, which reinforces the dual role of FSF as both a nutritional and structuring ingredient. Protein content also differed markedly between the seed ingredients. GSF showed low protein content (2.11%), whereas FSF exhibited a much higher value (27.34%), similar to that reported by Omode et al. [ 23 ] but lower than values described by Oderinde et al. [ 24 ] and Yusuf et al. [ 22 ]. In breads, protein contents ranged from 14.59% to 17.28%, all above the control (14.20%), with run 6 again presenting the highest level. This pattern agrees with studies demonstrating that protein-rich plant flours increase the protein content of breads [ 24 ]. Taken together, these results indicate that FSF functions as a protein-dense biomaterial capable of enriching breads without compromising gluten functionality, since dough expansion and loaf volume were maintained or improved. Crude fiber contents were low in GSF (0.67%) and higher in FSF (1.32%), although still slightly below values reported for C. pulcherrima seeds in some studies [ 22 , 24 ]. In the breads, crude fiber ranged from 0.23% to 0.53%, with run 6 presenting the highest value, whereas the other formulations did not differ significantly from the control (p > 0.05). These modest increases are consistent with the relatively low fiber content of FSF compared with classical high-fiber ingredients. Similar outcomes have been reported in breads formulated with moderate levels of plant-based ingredients, where fiber enrichment is limited by technological constraints [ 18 , 25 , 27 ]. FSF had a high ash content (3.85%), whereas GSF showed a much lower value (0.15%). Reported ash levels for C. pulcherrima seeds vary in the literature [ 22 – 24 ], but the present values confirm that the flour fraction concentrates most of the minerals. In breads, ash contents ranged from 1.62% to 2.97%, all above the control (1.57%), with run 6 presenting the highest value. These findings are in line with the Brazilian Food Composition Table and with studies showing that plant flours increase the mineral content of breads [ 18 , 27 – 28 ], reinforcing FSF as a nutritionally advantageous additive. Carbohydrate contents also reflected the distribution of macronutrients between the seed fractions. FSF exhibited a high carbohydrate content (51.66%), whereas GSF showed a much lower value (3.18%) (Table 3 ), confirming the predominance of carbohydrates in the flour fraction [ 22 , 24 – 25 ]. Bread carbohydrates ranged from 35.00% to 41.34%. Formulations 1, 2, 3, 4 and 7 were statistically similar to the control (p > 0.05), while the remaining formulations differed significantly, with run 5 presenting the highest value (41.34%) and run 6 the lowest (35.00%). These variations result from simultaneous changes in moisture, ash, protein, lipid and fiber contents, since carbohydrates were calculated by difference. Similar patterns have been reported in breads containing plant flours [ 8 , 18 , 25 ], indicating that FSF and GSF modify macronutrient distribution without deviating from typical ranges for pan bread. The response surface plots in Fig. 1 illustrate how FSF and GSF concentrations affect bread lipid and protein contents. Increasing FSF levels consistently raised both lipid and protein values, confirming FSF as the main contributor to the nutritional enrichment of the breads, whereas GSF had little effect within the evaluated range. Based on the significant regression coefficients (p < 0.05), the lipid content (Y LIPIDS ) was described by the following second-order model: \({Y}_{\left(LIPIDS\right)}\) = 6.99 + 0.41x 1 – 0.01x 1 2 + 0.23 x 2 – 0.0003x 2 2 – 0.02 x 1 x 2 (3) Where: x 1 = FSF (%), x 2 = GSF (%). The positive linear term for x₁ and the negative quadratic coefficient indicates that lipid content increases with FSF concentration up to the upper region of the experimental domain (10–11.04% FSF; x₁ = +1 to + 1.41). This trend is consistent with runs 3 (10% FSF, 1% GSF), 4 (10% FSF, 5% GSF) and 6 (11.04% FSF, 3% GSF), which presented the highest lipid contents (9.70–10.11%). In contrast, the non-significant or small coefficients for x₂ confirm that GSF did not markedly influence lipid content within the studied range (up to 5.83%). Protein content followed a similar pattern and was modelled as: Y (PROTEINS) = 13.20 + 0.29x 1 + 0.006x 1 2 + 0.05x 2 + 0.01x 2 2 − 0.01 x 1 x 2 (4) where: x 1 = FSF (%), x 2 = GSF (%). As shown in Fig. 1 , increasing FSF concentration resulted in a proportional rise in protein content. Formulations containing 10–11.04% FSF (x₁ = +1 to + 1.41) yielded the highest protein values, with runs 3, 4 and 6 reaching 16.41–17.28%. GSF concentration did not significantly affect protein content within the evaluated range. Together, these models highlight FSF as the key driver of nutritional enhancement, while GSF plays a minor role in macronutrient enrichment but contributes structurally via its hydrated galactomannan matrix. Figure 2 shows the SEM micrographs of FSF, GSF, the control bread crumb, and the enriched bread crumb (Run 4: 10% FSF, 5% GSF). The microstructural contrasts among these samples help clarify how FSF and GSF interact within the bread matrix and contribute to their functional effects in the formulations. The micrograph of FSF shows irregular particles with angular fragments, compact aggregates and rough surfaces, typical of protein- and fiber-rich seed flours. This morphology reflects the presence of galactomannans, proteins and lipids adhering to the matrix and suggests high water-binding capacity and interaction potential with gluten, explaining the improved dough viscoelasticity in formulations with higher FSF levels [ 29 ]. The GSF micrograph reveals a compact, sponge-like and cohesive gel, characteristic of hydrocolloid-rich systems. Its smooth, continuous structure indicates a three-dimensional galactomannan network with strong water retention and gas-cell stabilization capacity, supporting dough hydration without impairing gluten development and consistent with its high moisture and aw values. The control bread crumb exhibited a dense, compact starch–protein matrix with low porosity. Starch granules remained partially embedded in the protein phase, suggesting limited gelatinization and weaker structural integration. This morphology is consistent with a less stable gas-cell system and aligns with the lower specific volume, alveoli number and circularity observed [ 29 , 27 ]. In contrast, Run 4 (10% FSF, 5% GSF) showed a more cohesive, continuous crumb with fused starch domains, thinner cell walls and larger interconnected pores, indicating enhanced gelatinization and stronger interactions among FSF components (proteins, lipids, galactomannans) and gluten. This expanded, elastic microstructure explains the higher specific volume and improved alveoli characteristics [ 8 ]. Overall, the SEM results confirm that FSF reinforces the matrix through its protein, lipid and fiber fractions, whereas GSF contributes hydration and gas-cell stability. Together, they generate a more porous, homogeneous and mechanically stable crumb, consistent with the superior technological properties of the enriched breads. The CLSM image (Fig. 3) of FSF (A) shows a heterogeneous fluorescence pattern with red, blue and purple regions corresponding to proteins, polysaccharides and fiber-rich domains. This distribution reflects FSF’s nutritional density and its capacity for water binding and interaction with gluten, supporting the improved dough viscoelasticity in the enriched breads [ 30 ]. The GSF micrograph (B) presents a cohesive, uniformly fluorescent matrix typical of hydrocolloid gels, indicating a well-organized galactomannan network. This structure explains its high-water retention and contribution to gas-cell stabilization without disrupting gluten development. The control crumb (C) displayed a dense, poorly organized matrix with weak fluorescence and limited protein–starch definition, consistent with lower loaf volume and reduced aeration. This reflects the limited structural efficiency of the wheat matrix without added biomaterials [ 30 ]. In contrast, the enriched bread (D) exhibited a more open and continuous structure with clearer protein–starch interactions and more uniformly distributed fluorescent domains, supporting the improved macroscopic quality of the enriched breads. The confocal microscopy results confirm the synergistic action of FSF and GSF in breadmaking. FSF strengthens the matrix through its protein- and fiber-rich composition, whereas GSF enhances hydration and gas-cell stability. Together, they promote a more cohesive, elastic and aerated crumb, consistent with the improved physicochemical and technological attributes observed. 4. Conclusion This study demonstrated that galactomannan-rich ingredients obtained from Caesalpinia pulcherrima seeds, applied as seed flour (FSF) and seed gel (GSF), act as functional biomaterials in wheat bread, simultaneously modulating technological quality, nutritional composition and microstructure. Within the ranges evaluated using a central composite design, the incorporation of FSF and GSF increased loaf specific volume, enhanced crumb porosity and circularity, and maintained expansion index values comparable to the control. These effects indicate improved gas retention and a more aerated, homogeneous crumb without impairing dough expansion capacity. The proximate composition results showed that FSF is the main contributor to nutritional enrichment, leading to higher protein, lipid, ash and, to a lesser extent, fiber contents in enriched breads compared with the control. GSF, in turn, had minor impact on macronutrient levels but played a relevant structural role by contributing to a hydrated galactomannan matrix. Moisture and water activity values of the breads remained within typical ranges for pan bread and complied with regulatory limits, while pH and titratable acidity values confirmed that FSF and GSF are compatible with standard yeast fermentation and do not compromise product safety. Microstructural analyses by SEM and CLSM provided mechanistic support for the macroscopic and compositional findings. FSF reinforced the gluten–starch matrix through its protein-, lipid- and fiber-rich particles, whereas GSF contributed a cohesive hydrocolloid network with high water-binding and gas-cell stabilization capacity. Together, these ingredients generated a more continuous, cohesive and porous crumb structure, explaining the improved technological performance of the enriched breads and establishing clear structure–function relationships for C. pulcherrima seed biomaterials in bakery systems. Overall, the results highlight C. pulcherrima seeds as a promising and underutilized tropical resource for the development of nutritionally enhanced and structurally tailored breads using clean-label, plant-based ingredients. Future studies should address sensory acceptance, shelf-life behavior and process scaling, as well as the incorporation of FSF and GSF into other cereal-based products, to further explore their potential in sustainable and functional food design. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Ethics and consent to participate The collection of plant material used in this study complied with applicable institutional, local, and national guidelines and legislation. Pods/seeds of Caesalpinia pulcherrima (L.) Sw. (Fabaceae) were collected from cultivated ornamental trees located at different sites on the campus of the Federal University of Ceará (UFC), Fortaleza, Ceará, Brazil (Latitude: 3°44'38.6"S; Longitude: 38°34'47.3"W). The plant material was taxonomically identified by Prof. Dr. Maria Iracema Bezerra Loiola (Curator, Herbarium Prisco Bezerra – EAC). Voucher specimen(s) were deposited in the Herbarium Prisco Bezerra (EAC), Federal University of Ceará, Fortaleza, Ceará, Brazil, under accession number EAC 67246. This study did not involve human participants or animals; therefore, ethics approval and consent to participate are not applicable. Consent to publish Not applicable. Funding The authors did not receive support from any organization for the submitted work. Author Contribution Conceptualization: Rafael Audino Zambelli. Methodology: Neilane Gomes da Rocha, Elenilson Godoy Alves Filho. Investigation: Poliana Brito de Sousa, Neilane Gomes da Rocha, Camila de Carvalho Chaves. Formal analysis: Poliana Brito de Sousa, Camila de Carvalho Chaves. Resources: Ana Karoline Nogueira Freitas. Supervision: Ana Karoline Nogueira Freitas, Rafael Audino Zambelli. Validation: Elenilson Godoy Alves Filho. Project administration: Rafael Audino Zambelli. Writing – original draft: Poliana Brito de Sousa. Writing – review and editing: Elenilson Godoy Alves Filho, Rafael Audino Zambelli. Acknowledgement The authors thank IFPI, IFMA and Embrapa Meio-Norte for support and access to laboratory facilities used in this work. We also acknowledge the Federal University of Ceará and the Analytical Center-UFC/CT-INFRA/MCTI-SISNANO/Pro-Equipment CAPES, which contributed to the success of this study. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. References L.C.N.M. Bezerra, C.E.M. Silva, H.O. Nascimento, P.B.L. Constant, Produção e caracterização de pão de forma obtido por processo rápido. Res. Soc. Dev. 11 (11), e297111133569 (2022). https://doi.org/10.33448/rsd-v11i11.33569 A. Purkiewicz, F.H. Gul, R. Pietrzak-Fiecko, The utilization of vegetable powders for bread enrichment: Effect on selected minerals and phenolic content. Appl. Sci. 14 , 10022 (2024). https://doi.org/10.3390/app142110022 A.M. Gheno, J.P. Geadicke, L. Müller, F. Stoffel, Avaliação de atributos tecnológicos de pão francês com adição de farinhas de vegetais. Braz. J. Food Technol. 25 , e2021113 (2022). https://doi.org/10.1590/1981-6723.11321 F.C.A. Buriti, K.M.O. Santos, V.G. Sombra, J.S. Maciel, D.M.A. Teixeira Sá, H.O. Salles, G. Oliveira, R.C.M. Paula, J.P.A. Feitosa, A.C.O.M. Moreira, R.A. Moreira, A.S. Egito, Characterisation of partially hydrolysed galactomannan from Caesalpinia pulcherrima seeds as a potential dietary fibre. Food Hydrocoll. 35 , 512–521 (2014). https://doi.org/10.1016/j.foodhyd.2013.07.015 A.A.C. Passos, M. Lovera, M.S.R. Bastos, J.S. Maciel, V.G. Sombra, R.C. Braga, A.C.O.M. Moreira, R.M. Moreira, Low-viscosity dietary fiber production by enzymatic hydrolysis of galactomannan from Caesalpinia pulcherrima seeds: Optimization and physicochemical characterization. J. Food Process. Preserv. 45 , e15949 (2021). https://doi.org/10.1111/jfpp.15949 S. Senarathna, S. Navaratne, I. Wickramasinghe, R. Coorey, Development and characterization of Caesalpinia pulcherrima seed gum-based films for food packaging. J. Consumer Prot. Food Saf. 17 , 65–72 (2022). https://doi.org/10.1007/s00003-021-01347-9 D.J.S. Guimarães, R.A. Zambelli, M.R.A. Afonso, D.F. Pontes, Cryoprotective potential of vegetable powders and polydextrose in frozen bread doughs. Int. J. Refrig. 172 , 108–119 (2025). https://doi.org/10.1016/j.ijrefrig.2025.01.010 R.A. Zambelli, L.I.F. Pinto, S. Junior, E.C. Lima, A.C.V. Goiana, M. L., L.G. Mendonça, Effect of black sesame and flaxseed flour on bread quality using response surface methodology. J. Eng. Process. Manage. 10 (2), 32–40 (2018). https://doi.org/10.7251/jepm181002032z American Association of Cereal Chemists, Approved methods of the American Association of Cereal Chemists , 10th edn. (AACC International, 2000) U. Gonzales-Barrón, F. Butler, A comparison of seven thresholding techniques with the k-means clustering algorithm for measurement of bread-crumb features by digital image analysis. J. Food Eng. 74 , 268–278 (2006). https://doi.org/10.1016/j.jfoodeng.2005.03.007 Instituto Adolfo Lutz, Métodos físico-químicos para análise de alimentos , 4– edn. (ed.). Instituto Adolfo Lutz, 2008) Association of Official Analytical Chemists, Official methods of analysis , 16th edn. (AOAC International, 2005) A.S. Abdel-Ghany, D.A. Zaki, Evaluation of technological and antihyperglycemic effects of pan bread enriched with okra pod waste on diabetic male rats. Alexandria Sci. Exch. J. 44 , 691–708 (2023). https://doi.org/10.21608/asejaiqjsae.2023.329465 A. Wirkijowska, P. Zarzycki, A. Sobota, A. Nawrocka, A. Blicharz-Kania, D. Andrejko, The possibility of using by-products from the flaxseed industry for functional bread production. LWT. 118 , 108860 (2020). https://doi.org/10.1016/j.lwt.2019.108860 T. Lafarga, E. Gallagher, R.E. Aluko, M.A.E. Auty, M. Hayes, Addition of an enzymatic hydrolysate of bovine globulins to bread and determination of hypotensive effects in spontaneously hypertensive rats. J. Agric. Food Chem. 64 , 1741–1750 (2016). https://doi.org/10.1021/acs.jafc.5b06078 de L.G. Mendonça, M.H.B. Holanda, M.V.S. Leão, A.M.M. Theóphilo Galvão, S.V. Carneiro, D.J.S. Guimarães, Assessing the impact of Plectranthus amboinicus leaf extract on bread quality. Cereal Chem. 101 (2) (2024). https://doi.org/10.1002/cche.10794 C.M. Ferreira, S.B. Lima, R.A. Zambelli, M.R.A. Afonso, Efeito da farinha mista de subprodutos vegetais em pães tipo forma. Brazilian J. Dev. 6 (2), 8710–8724 (2020). https://doi.org/10.34117/bjdv6n2-254 R.A. Zambelli, A.M.T. Galvão, L.I.F. Pinto, G.B.M. Santos, A.C. Silva, C.A.L. Melo, M.M. Farias, L.G. Mendonça, Effect of vegetable powders on the bread quality made from frozen dough. Int. J. Nutr. Food Sci. 6 (6–1), 1–8 (2017). https://doi.org/10.11648/j.ijnfs.s.2017060601.11 R.S. Selvi, S. Gopalakrishanan, M. Ramajayam, R. Soman, Evaluation of mucilage of Caesalpinia pulcherrima as binder for tablets. Int. J. ChemTech Res. 2 (1), 436–442 (2010) B.D.G. Franco, M. Landgraf, (2004). Microbiologia dos alimentos . Atheneu C.G. Rizzello, M. Verni, S. Bordignon, V. Gramaglia, M. Gobbetti, Hydrolysate from a mixture of legume flours with antifungal activity as an ingredient for prolonging the shelf-life of wheat bread. Food Microbiol. 64 , 72–82 (2017). https://doi.org/10.1016/j.fm.2016.12.003 A.A. Yusuf, B.M. Mofio, A.B. Ahmed, Nutrient contents of Caesalpinia pulcherrima seeds. Pakistan J. Nutr. 6 (2), 117–121 (2007) A.A. Omode, O.S. Fatoki, K.A. Olaogun, Physicochemical properties of some underexploited and nonconventional oilseeds. J. Agric. Food Chem. 43 , 2850–2853 (1995). https://doi.org/10.1021/jf00059a015 R.A. Oderinde, A. Adewuyi, I.A. Ajayi, Determination of mineral nutrients, characterization and analysis of fat-soluble vitamins of Caesalpinia pulcherrima and Albizia lebbeck seeds and seed oils. Seed Sci. Biotechnol. 2 (2), 74–78 (2008) M.O. Aljobair, Enrichment of bread with green pumpkin, watermelon and cucumber peels: Physicochemical, pasting, rheological, antioxidant and organoleptic properties. J. Food Qual. 2024 (2024). Article 6649325. https://doi.org/10.1155/2024/6649325 S. Kumar, S. Arora, V. Kumar, S. Joshi, B. Naik, B. Bisht, M.S. Tomar, P. Gururani, Physicochemical, nutritional and sensory characteristics of Chenopodium album , ashwagandha, flaxseed and giloy fortified bun. J. Food Process. Preserv. 46 , e17265 (2022). https://doi.org/10.1111/jfpp.17265 L. Menon, S.D. Majumdar, U. Ravi, Development and analysis of composite flour bread. J. Food Sci. Technol. 52 (7), 4156–4165 (2015). https://doi.org/10.1007/s13197-014-1466-8 C. Li, G. Chen, M. Tilley, R. Chen, M. Perez-Fajardo, X. Wu, Y. Li, Enhancing gluten network formation and bread-making performance of wheat flour using wheat bran aqueous extract. Foods. 13 (10), 1479 (2024). https://doi.org/10.3390/foods13101479 H.A. Rathnayake, S.B. Navaratne, C.M. Navaratne, Porous crumb structure of leavened baked products. Int. J. Food Sci. 2018 , 8187318 (2018). https://doi.org/10.1155/2018/8187318 G. Scott, J.M. Awika, Effect of protein–starch interactions on starch retrogradation and implications for food product quality. Compr. Rev. Food Sci. Food Saf. 22 (3), 2081–2111 (2023). https://doi.org/10.1111/1541-4337.13141 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Mar, 2026 Reviews received at journal 08 Mar, 2026 Reviewers agreed at journal 03 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviewers agreed at journal 03 Mar, 2026 Reviewers invited by journal 03 Mar, 2026 Editor assigned by journal 02 Mar, 2026 Submission checks completed at journal 02 Mar, 2026 First submitted to journal 26 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8977729","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601496960,"identity":"12f30042-70d3-4740-ad89-648f51dbd152","order_by":0,"name":"Poliana Brito de Sousa","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Poliana","middleName":"Brito","lastName":"de Sousa","suffix":""},{"id":601496961,"identity":"95ce520b-c521-42ff-8eb2-b9609cc43e2d","order_by":1,"name":"Neilane Gomes da Rocha","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Neilane","middleName":"Gomes da","lastName":"Rocha","suffix":""},{"id":601496962,"identity":"0fb9c14b-4809-4a14-aa8d-4e5586e8dd26","order_by":2,"name":"Camila de Carvalho Chaves","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"de Carvalho","lastName":"Chaves","suffix":""},{"id":601496963,"identity":"66014fdf-238e-4d50-920d-aa1a3b2e54f9","order_by":3,"name":"Ana Karoline Nogueira Freitas","email":"","orcid":"","institution":"Federal Institute of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Karoline Nogueira","lastName":"Freitas","suffix":""},{"id":601496964,"identity":"678708b0-d92b-4556-85b8-09847d3b6614","order_by":4,"name":"Elenilson Godoy Alves Filho","email":"","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":false,"prefix":"","firstName":"Elenilson","middleName":"Godoy Alves","lastName":"Filho","suffix":""},{"id":601496965,"identity":"3df7d666-848a-4a33-a69a-84bb19d5d600","order_by":5,"name":"Rafael Audino Zambelli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYHACNgaGAgsefgbGBwyMDURrMZDgkWxgNiBNC4PBAWK1yLefTnvwwUBCxvhGMuMHxh33CGth7MndbjgD6DCzG8nMEoxniglrYWbI3SbNA9aSf4yBsS2BsBY2/rfbpP8AtRjPSGYjTguPBNAWUIgZSBCrRULi7XbDHqAWiTOPmSUSzxChRb4/d9uDHxU29vztwBD7uIMILaiAZA2jYBSMglEwCrADAJCoL/qmUmz/AAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Ceará","correspondingAuthor":true,"prefix":"","firstName":"Rafael","middleName":"Audino","lastName":"Zambelli","suffix":""}],"badges":[],"createdAt":"2026-02-26 12:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8977729/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8977729/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104258159,"identity":"b98ed58c-e700-4ea0-9634-70fe526ae130","added_by":"auto","created_at":"2026-03-09 17:36:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":224036,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface plots showing the effects of FSF and GSF levels on (a) lipid and (b) protein contents of breads.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8977729/v1/bc79ac2ea1b01fdb1a63e20d.png"},{"id":104258160,"identity":"75efd1f4-dd03-4c85-aed6-65c3f9eca552","added_by":"auto","created_at":"2026-03-09 17:36:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":670496,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of FSF, GSF, and the bread crumb samples enriched with these ingredients. (A) - FSF; (B) - GSF; (C) - Control bread; (D) – Bread enriched with 10% FSF and 5% GSF (Run 4).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8977729/v1/96974077ad1d040a73f465f5.png"},{"id":104258161,"identity":"85ae8640-d453-44f2-a8ee-21fe1401415f","added_by":"auto","created_at":"2026-03-09 17:36:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":708169,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning micrographs of \u003cem\u003eCaesalpinia pulcherrima\u003c/em\u003e seed flour (FSF), seed gel (GSF), and the crumb of breads enriched with these ingredients. (A) – FSF; (B) – GSF; (C) – Control bread; (D) – Run 4 (10% FSF, 5% GSF).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8977729/v1/ca9397b2aef467bc1a9c2d79.png"},{"id":104404971,"identity":"978a9536-4ada-41cc-91cd-eb188d85fdca","added_by":"auto","created_at":"2026-03-11 12:21:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2727035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8977729/v1/36877a53-0bd3-40ca-8c05-4dd40d951745.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Galactomannan-rich ingredients from Caesalpinia pulcherrima seeds reorganize wheat bread microstructure and enhance technological and nutritional quality","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBread is one of the most consumed staple foods worldwide, typically eaten one to three times a day depending on cultural habits and cereal cost. Its production follows standardized steps, mixing, gluten development, fermentation, baking, and packaging. Although designed to meet basic energy needs, consumer demand increasingly favors foods that support health and prevent diet-related diseases. Conventional wheat bread, however, is largely composed of refined carbohydrates and contains limited protein, fiber, minerals and vitamins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA common strategy to improve nutritional quality is the partial replacement of wheat flour with plant-based ingredients such as whole grains, legume flours, by-products or natural hydrocolloids. Yet, due to their gluten-free and fiber-rich nature, these ingredients often impair dough rheology, gas retention and loaf volume when used at high levels [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Legume-derived materials are particularly attractive because they combine proteins, lipids, minerals and dietary fiber with polysaccharides capable of modifying the dough matrix.\u003c/p\u003e \u003cp\u003eAmong tropical legumes, \u003cem\u003eCaesalpinia pulcherrima\u003c/em\u003e, known as flamboyant-mirim, dwarf poinciana or Barbados pride, is widely distributed in tropical regions such as Brazil, India, Malaysia and Sri Lanka and is well adapted to the Brazilian Cerrado. Various plant parts exhibit biological activities [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIts seeds are rich in galactomannans, soluble dietary fiber with thickening, stabilizing and film-forming properties [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These polysaccharides are applied in dairy desserts, edible films and fiber-enriched foods and strongly influence water binding, viscosity and phase behavior. In bread, galactomannans can interact with gluten proteins and starch granules, modulating gas cell stability, crumb structure and staling. Additionally, \u003cem\u003eC. pulcherrima\u003c/em\u003e seed flour has been explored for biofuel and animal feed due to its protein- and lipid-rich matrix, suggesting nutritional density coupled with functional macromolecules.\u003c/p\u003e \u003cp\u003eDespite these attributes, no studies have evaluated \u003cem\u003eC. pulcherrima\u003c/em\u003e seed derivatives in breadmaking. This knowledge gap represents an opportunity to (i) valorize an underutilized tropical legume; (ii) investigate how its galactomannan-rich biomaterials interact with the gluten\u0026ndash;starch network; and (iii) establish structure\u0026ndash;function relationships relevant to enriched bakery products.\u003c/p\u003e \u003cp\u003eTherefore, galactomannan-rich ingredients from underutilized tropical legumes such as Caesalpinia pulcherrima represent an attractive strategy to design bakery products with improved structure and nutritional quality. However, the combined effects of seed flour and gel forms on dough behavior, bread technological performance and microstructure remain unexplored. In this context, the present study aimed to elucidate the structure\u0026ndash;function relationships of \u003cem\u003eC. pulcherrima\u003c/em\u003e seed flour (FSF) and gel (GSF) in wheat bread, using response surface methodology to optimize their levels and advanced imaging techniques (SEM and CLSM) to link microstructural organization with technological and nutritional attributes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Raw material collection\u003c/h2\u003e \u003cp\u003ePods of \u003cem\u003eCaesalpinia pulcherrima\u003c/em\u003e containing mature seeds were collected at the Federal University of Cear\u0026aacute;, Campus Pici, Fortaleza, Brazil (3\u0026deg;44\u0026prime;38.6\u0026Prime;S; 38\u0026deg;34\u0026prime;47.3\u0026Prime;W), under SisGen authorization ABF331B. Pods were selected based on dry texture, natural dehiscence, and brown coloration. A voucher specimen was deposited in the Prisco Bezerra Herbarium (EAC 67246).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of \u003cem\u003eC. pulcherrima\u003c/em\u003e seed flour (FSF)\u003c/h2\u003e \u003cp\u003ePods were manually opened, and seeds were selected for integrity and absence of defects. Seeds were ground in an industrial blender (Li1.5, Skymsen, Brazil) for 5 min in 1-min intervals and then milled in a knife mill (Luca-226/5, Lucadema, Brazil) for 5 min under the same conditions. The powder was sieved through a 0.250 mm mesh using an electromagnetic shaker (Rotachoc Chopin\u0026reg;, France) for 10 min. The resulting seed flour (FSF; 0.250 mm) was vacuum-packed, stored at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of \u003cem\u003eC. pulcherrima\u003c/em\u003e seed gel (GSF)\u003c/h2\u003e \u003cp\u003eFor the preparation of GSF, 20 g of seeds were mixed with 200 mL of distilled water and heated on a magnetic hot plate (Q310-22B, Quimis, Brazil) at 70\u0026deg;C for 5 h. After cooling to room temperature, the cooked mixture was homogenized with a vertical mixer (Black \u0026amp; Decker, Brazil) and filtered through a fine mesh sieve to obtain the gel. The GSF was stored in polyethylene containers at 4\u0026deg;C (Electrolux, Brazil). The gel yield was approximately 69.7%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Bread formulation and experimental design\u003c/h2\u003e \u003cp\u003eThe control bread formulation, without FSF or GSF, followed Guimar\u0026atilde;es et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and consisted of wheat flour (100%), water (58%), hydrogenated vegetable fat (10%), refined sugar (5%), instant dry yeast (3.3%) and salt (2%). Ingredients were purchased from a local market in Teresina, Piau\u0026iacute;, Brazil.\u003c/p\u003e \u003cp\u003eThe experimental design consisted of eleven runs combining different concentrations of FSF and GSF according to the Central Composite Rotational Design. Run 1 was formulated with low levels of both factors (X₁ = \u0026minus;1; 5.0% FSF and X₂ = \u0026minus;1; 1.0% GSF), while Run 2 maintained the same FSF level (5.0%) combined with the high GSF level (X₂ = +1; 5.0%). Runs 3 and 4 used the high FSF concentration (X₁ = +1; 10.0%), combined with low GSF (1.0%) in Run 3 and high GSF (5.0%) in Run 4. The axial points included Run 5 (X₁ = \u0026minus;1.41; 3.96% FSF and X₂ = 0; 3.0% GSF) and Run 6 (X₁ = +1.41; 11.04% FSF and X₂ = 0; 3.0% GSF). Runs 7 and 8 were formulated with the central FSF level (X₁ = 0; 7.5%) combined with the lowest axial GSF level (X₂ = \u0026minus;1.41; 0.17%) in Run 7 and the highest axial GSF level (X₂ = +1.41; 5.82%) in Run 8. Finally, Runs 9, 10 and 11 corresponded to the central point of the design, containing 7.5% FSF and 3.0% GSF in all replicates (X₁ = 0; X₂ = 0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Bread-making process\u003c/h2\u003e \u003cp\u003eDough ingredients were mixed in a planetary mixer (Arno, Brazil) for 7 min, divided into 250 g portions and manually shaped. Loaves were placed in single-sheet pans and proofed at 32\u0026deg;C and 75% RH for 90 min in a fermentation chamber (Frilux, Brazil). Baking was performed in an electric oven (Ven\u0026acirc;ncio, Brazil) at 220\u0026deg;C for 20 min, followed by cooling at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C). Bread quality analyses were conducted 2 h after baking [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Expansion index (EI)\u003c/h2\u003e \u003cp\u003eThe EI was determined according to Zambelli et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Cylindrical dough portions (~\u0026thinsp;10 g) had their diameter and height measured with a calibrated caliper before fermentation and after baking. EI was calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$EI=\\frac{({D}_{P}/{H}_{P})}{({D}_{M}/{H}_{M})}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere:\u003c/p\u003e \u003cp\u003e \u003cem\u003eDₚ\u003c/em\u003e \u0026ndash; diameter of the bread after baking; \u003cem\u003eHₚ\u003c/em\u003e \u0026ndash; height of the bread after baking; \u003cem\u003eDₘ\u003c/em\u003e \u0026ndash; diameter of the molded dough before fermentation; \u003cem\u003eHₘ\u003c/em\u003e \u0026ndash; height of the molded dough before fermentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Specific volume of bread\u003c/h2\u003e \u003cp\u003eBread volume was measured by the millet seed displacement method (AACC Method 72\u0026thinsp;\u0026minus;\u0026thinsp;10) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] in triplicate. Specific volume (mL.g⁻\u0026sup1;) was calculated as the ratio between bread volume and weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Crumb structure image analysis\u003c/h2\u003e \u003cp\u003eThe crumb structure was analyzed by digital image processing. Central bread slices were scanned at 550 dpi using an HP ScanJet 2400, and images (900 \u0026times; 900 pixels) were processed in ImageJ\u0026reg; 1.47v. Images were converted to 8-bit grayscale, thresholder using Otsu\u0026rsquo;s method, and analyzed for alveoli number and circularity following [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Physicochemical and proximate analyses\u003c/h2\u003e \u003cp\u003ePhysicochemical analyses (pH, titratable acidity, protein, moisture, ash, and crude fiber) were performed for FSF, GSF, and bread samples according to Adolfo Lutz Institute [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and AOAC [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Water activity (aw) was measured using a LabSwift-aw analyzer (Novasina, Switzerland), and carbohydrate content was calculated by difference. All determinations were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Scanning Electron Microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThe microstructure of FSF, GSF, and bread crumb samples was examined by SEM. Samples were fixed on aluminum stubs with carbon tape and sputter-coated with gold (~\u0026thinsp;20 nm) using a Bal-Tec SCD 050 coater. Micrographs were obtained in a JEOL JSM-6390LV microscope operated at 15 kV, with magnifications between 500\u0026times; and 2000\u0026times; to visualize surface morphology, starch granules, and the protein\u0026ndash;starch network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Confocal Laser Scanning Microscopy (CLSM)\u003c/h2\u003e \u003cp\u003eCLSM was used to visualize the crumb microstructure and the distribution of starch and protein. Samples were mounted on glass slides and analyzed using a Zeiss LSM 710 microscope. Rhodamine B (543/560\u0026ndash;620 nm) and FITC (488/500\u0026ndash;540 nm) were used to label protein and starch, respectively. Images were acquired using ZEN software and processed to assess the three-dimensional organization and interactions within the protein\u0026ndash;starch matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences among treatments, control bread, and raw materials were assessed by ANOVA followed by Tukey\u0026rsquo;s test at 5% significance using Statistica\u0026reg; 10.0 (StatSoft Inc., USA). The effects of FSF (x₁) and GSF (x₂) on bread quality variables were modeled using second-order polynomial regression, as shown in Eq.\u0026nbsp;(2).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;β\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;β\u003csub\u003e1\u003c/sub\u003ex\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;β\u003csub\u003e2\u003c/sub\u003ex\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;β\u003csub\u003e11\u003c/sub\u003ex\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;β\u003csub\u003e22\u003c/sub\u003ex\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;β\u003csub\u003e12\u003c/sub\u003e x\u003csub\u003e1\u003c/sub\u003e x\u003csub\u003e2\u003c/sub\u003e (2)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eY\u003c/em\u003e is the dependent variable; \u003cem\u003eβ₀\u003c/em\u003e is the intercept; \u003cem\u003eβ₁\u003c/em\u003e and \u003cem\u003eβ₂\u003c/em\u003e are the linear coefficients; \u003cem\u003eβ₁₁\u003c/em\u003e and \u003cem\u003eβ₂₂\u003c/em\u003e are the quadratic coefficients; and \u003cem\u003eβ₁₂\u003c/em\u003e represents the interaction coefficient between the independent variables.\u003c/p\u003e \u003cp\u003eModel significance was assessed using the F-test, lack-of-fit, and determination coefficients (R\u0026sup2;). Validated models were then used to generate response surface and contour plots, illustrating how FSF and GSF levels influenced the technological and nutritional attributes of the breads.\u003c/p\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eThe experimental values obtained for alveoli circularity and count, specific volume and expansion index are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, together with the corresponding measurements of the control bread for comparison.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026ndash; Mean values and Tukey\u0026rsquo;s test results for technological properties of breads supplemented with FSF and GSF.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRuns\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlveoli circularity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlveoli number\u003c/p\u003e\n \u003cp\u003e(cells)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecific Volume (mL.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.928\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1034\u0026thinsp;\u0026plusmn;\u0026thinsp;58.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.900\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1120\u0026thinsp;\u0026plusmn;\u0026thinsp;45.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.903\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1199\u0026thinsp;\u0026plusmn;\u0026thinsp;38.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.939\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1244\u0026thinsp;\u0026plusmn;\u0026thinsp;29.00\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.832\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e912\u0026thinsp;\u0026plusmn;\u0026thinsp;31.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.952\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1383\u0026thinsp;\u0026plusmn;\u0026thinsp;19.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.908\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1093\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.898\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1012\u0026thinsp;\u0026plusmn;\u0026thinsp;32.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.910\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1183\u0026thinsp;\u0026plusmn;\u0026thinsp;14.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.905\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1179\u0026thinsp;\u0026plusmn;\u0026thinsp;18.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.908\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1190\u0026thinsp;\u0026plusmn;\u0026thinsp;21.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.639\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e728\u0026thinsp;\u0026plusmn;\u0026thinsp;30.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent lowercase letters in the same column indicate significant differences according to Tukey\u0026rsquo;s test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eThe incorporation of FSF and GSF increased the specific volume of all enriched breads compared with the control, indicating improved gas retention during proofing and baking. The highest specific volume (3.87 mL g⁻\u0026sup1;) was observed in run 6 (11.04% FSF\u0026thinsp;+\u0026thinsp;3% GSF), whereas the control showed the lowest value (2.16 mL g⁻\u0026sup1;). Crumb image analysis further supported these macroscopic differences: breads containing FSF and GSF had higher alveoli numbers (1034\u0026ndash;1383) and circularity (0.832\u0026ndash;0.952) than the control (728; 0.639), resulting in a more homogeneous crumb with well-distributed and rounded gas cells [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite these structural changes, the expansion index (EI) remained statistically unchanged (1.24\u0026ndash;1.35 cm; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that the partial replacement of wheat flour with FSF and GSF preserved dough expansion capacity while allowing higher loaf volume and a finer crumb structure.\u003c/p\u003e\n\u003cp\u003eThe observed increases in loaf volume and crumb aeration are mainly related to the galactomannans naturally present in the seeds. These soluble fibers and hydrocolloids exhibit high water-holding capacity, increase dough viscosity and stabilize gas cells during fermentation. In gluten-containing systems, galactomannans interact with gluten proteins and starch granules, reinforcing the dough matrix and promoting higher loaf volume [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition to the polysaccharide effect, the slightly higher lipid contents measured in FSF- and GSF-enriched breads (8.78\u0026ndash;10.11%) compared with the control (8.28%) may also have contributed to this behavior. Lipids can enhance dough aeration and stabilize gas bubbles by forming surface-active complexes with proteins and starch, strengthening gas-cell walls during proofing and baking.\u003c/p\u003e\n\u003cp\u003eComparable improvements in loaf volume and crumb uniformity have been reported in breads enriched with plant-based flours or hydrocolloids, which modify dough rheology and gas retention [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e] In line with these studies, the present results reinforce that galactomannan-rich legume ingredients behave as functional biomaterials capable of strengthening the gluten\u0026ndash;starch network and improving bread structure. From a biomaterials standpoint, FSF and GSF therefore act as structuring agents that generate a more cohesive and mechanically stable wheat-based crumb, explaining the improved technological performance observed.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026ndash; pH, titratable acidity, moisture, and water activity of breads with FSF and GSF, control, and raw materials.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRuns\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTitratable acidity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMoisture (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWater Activity\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n 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\u003cp\u003e34.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.954\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.943\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.951\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.950\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ecdef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.950\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.948\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.951\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFSF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.496\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.986\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent lowercase letters in the same column indicate significant differences according to Tukey\u0026rsquo;s test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eThe experimental results for pH, titratable acidity, moisture and water activity are presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e for the control bread, FSF, GSF and enriched breads. FSF and GSF showed slightly acidic pH values (6.29 and 6.25, respectively), which are typical of bakery ingredients and within the range reported for \u003cem\u003eC. pulcherrima\u003c/em\u003e seed mucilage (pH 5.7) [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although near-neutral pH foods may favor spoilage microorganism growth [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], the pH values observed for FSF and GSF remain compatible with safe incorporation into bread formulations.\u003c/p\u003e\n\u003cp\u003eThe pH of breads containing FSF and GSF ranged from 5.40 to 5.55, while the control presented a slightly lower value (5.38). Formulations 1\u0026ndash;3 (5.40\u0026ndash;5.43) were similar to the control, whereas runs 4\u0026ndash;11 exhibited higher pH values. This range is technologically adequate, since yeast fermentation is optimal between pH 4.5 and 6.5 and conventional breads typically show pH 4.7\u0026ndash;5.4 due to organic acid formation during fermentation [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Previous studies have also reported only minor pH changes when plant-based ingredients are incorporated into wheat doughs [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], indicating that FSF and GSF slightly increased pH without impairing fermentation.\u003c/p\u003e\n\u003cp\u003eTitratable acidity, which is related to freshness and shelf stability [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e], was lowest in FSF and GSF (0.12%) and ranged from 0.16% to 0.30% in breads. Among the breads, formulation 2 exhibited the lowest acidity, whereas the control showed the highest value, in agreement with reports of reduced acidity when plant ingredients partially replace wheat flour [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The lower acidity of enriched breads, combined with essentially unchanged water activity values (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), suggests that the observed differences were driven by the intrinsic composition of FSF and GSF rather than by deviations in fermentation conditions, supporting process reproducibility and compatibility with standard breadmaking.\u003c/p\u003e\n\u003cp\u003eMoisture values reflected the contrasting nature of the raw materials. FSF showed low moisture (5.92%), whereas GSF presented very high moisture (95.24%), in agreement with previous reports for \u003cem\u003eC. pulcherrima\u003c/em\u003e seeds and mucilage [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. FSF complied with Brazilian standards for flours (maximum 15% moisture; RDC 263/2005), confirming its suitability as a dry ingredient. Bread moisture ranged from 32.33% to 37.09%, with the control displaying the highest value and formulation 4 (10% FSF and 5% GSF) the lowest. These differences can be attributed to the balance between the additional water introduced with GSF, the water-binding capacity of FSF and evaporative losses during baking. All breads met regulatory limits for pan bread moisture (\u0026le;\u0026thinsp;38%; RDC 90/2000), indicating that FSF and GSF did not compromise product safety or legal compliance. Although several studies have reported increased moisture when plant-based ingredients are incorporated into breads [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], the present results show that, under the conditions tested, FSF and GSF levels were compatible with moisture contents typical of conventional breads.\u003c/p\u003e\n\u003cp\u003eWater activity followed a complementary pattern. FSF exhibited low aw (0.496), whereas GSF showed a very high value (0.986), which explains the need for refrigeration of the gel. Bread aw ranged from 0.943 to 0.956, without significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) relative to the control. These values confirm that all breads are high-aw products, which accounts for their naturally short shelf life and the need for appropriate packaging and storage conditions. The lack of significant changes in aw after FSF and GSF incorporation indicates that the microenvironment of available water in the crumb was not markedly altered, despite differences in formulation and moisture. Similar trends of essentially unchanged aw in enriched breads have been reported when plant-based ingredients are added to wheat-based formulations [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe proximate composition results (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) highlighted marked differences in lipid content between the seed-derived ingredients. GSF had a very low lipid content (0.08%), whereas FSF exhibited a substantially higher value (9.90%), consistent with previous reports for \u003cem\u003eC. pulcherrima\u003c/em\u003e seeds [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. This lipid fraction is intrinsic to the seed and, when transferred to the flour, represents a natural source of fats for bakery formulations, offering the possibility of improving fatty acid profiles compared with conventional breads.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026ndash; Proximate composition and Tukey\u0026rsquo;s test for breads with FSF and GSF, control, and raw materials.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRuns\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLipids\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProteins\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCrude Fiber (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarbohydrate (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ecdef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003efgh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003efgh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003edefg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent lowercase letters in the same column indicate significant differences according to Tukey\u0026rsquo;s test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eIn the breads, lipid contents ranged from 8.78% to 10.11%, while the control presented the lowest value (8.28%). The highest lipid level was observed in run 6 (11.04% FSF\u0026thinsp;+\u0026thinsp;3% GSF), mirroring the higher FSF inclusion. Similar increases in lipid content have been reported in breads enriched with plant flours [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. From a technological perspective, these lipids interact with gluten and starch, improving gas retention, loaf volume and crumb elasticity, which reinforces the dual role of FSF as both a nutritional and structuring ingredient.\u003c/p\u003e\n\u003cp\u003eProtein content also differed markedly between the seed ingredients. GSF showed low protein content (2.11%), whereas FSF exhibited a much higher value (27.34%), similar to that reported by Omode et al. [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] but lower than values described by Oderinde et al. [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] and Yusuf et al. [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. In breads, protein contents ranged from 14.59% to 17.28%, all above the control (14.20%), with run 6 again presenting the highest level. This pattern agrees with studies demonstrating that protein-rich plant flours increase the protein content of breads [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Taken together, these results indicate that FSF functions as a protein-dense biomaterial capable of enriching breads without compromising gluten functionality, since dough expansion and loaf volume were maintained or improved.\u003c/p\u003e\n\u003cp\u003eCrude fiber contents were low in GSF (0.67%) and higher in FSF (1.32%), although still slightly below values reported for \u003cem\u003eC. pulcherrima\u003c/em\u003e seeds in some studies [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the breads, crude fiber ranged from 0.23% to 0.53%, with run 6 presenting the highest value, whereas the other formulations did not differ significantly from the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These modest increases are consistent with the relatively low fiber content of FSF compared with classical high-fiber ingredients. Similar outcomes have been reported in breads formulated with moderate levels of plant-based ingredients, where fiber enrichment is limited by technological constraints [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eFSF had a high ash content (3.85%), whereas GSF showed a much lower value (0.15%). Reported ash levels for \u003cem\u003eC. pulcherrima\u003c/em\u003e seeds vary in the literature [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e], but the present values confirm that the flour fraction concentrates most of the minerals. In breads, ash contents ranged from 1.62% to 2.97%, all above the control (1.57%), with run 6 presenting the highest value. These findings are in line with the Brazilian Food Composition Table and with studies showing that plant flours increase the mineral content of breads [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], reinforcing FSF as a nutritionally advantageous additive.\u003c/p\u003e\n\u003cp\u003eCarbohydrate contents also reflected the distribution of macronutrients between the seed fractions. FSF exhibited a high carbohydrate content (51.66%), whereas GSF showed a much lower value (3.18%) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming the predominance of carbohydrates in the flour fraction [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Bread carbohydrates ranged from 35.00% to 41.34%. Formulations 1, 2, 3, 4 and 7 were statistically similar to the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the remaining formulations differed significantly, with run 5 presenting the highest value (41.34%) and run 6 the lowest (35.00%). These variations result from simultaneous changes in moisture, ash, protein, lipid and fiber contents, since carbohydrates were calculated by difference. Similar patterns have been reported in breads containing plant flours [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], indicating that FSF and GSF modify macronutrient distribution without deviating from typical ranges for pan bread.\u003c/p\u003e\n\u003cp\u003eThe response surface plots in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e illustrate how FSF and GSF concentrations affect bread lipid and protein contents. Increasing FSF levels consistently raised both lipid and protein values, confirming FSF as the main contributor to the nutritional enrichment of the breads, whereas GSF had little effect within the evaluated range. Based on the significant regression coefficients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the lipid content (Y\u003csub\u003eLIPIDS\u003c/sub\u003e) was described by the following second-order model:\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\({Y}_{\\left(LIPIDS\\right)}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e = 6.99\u0026thinsp;+\u0026thinsp;0.41x\u003csub\u003e1\u003c/sub\u003e \u0026ndash; 0.01x\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;0.23 x\u003csub\u003e2\u003c/sub\u003e \u0026ndash; 0.0003x\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e \u0026ndash; 0.02 x\u003csub\u003e1\u003c/sub\u003ex\u003csub\u003e2\u003c/sub\u003e (3)\u003c/p\u003e\n\u003cp\u003eWhere: x\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;FSF (%), x\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;GSF (%).\u003c/p\u003e\n\u003cp\u003eThe positive linear term for x₁ and the negative quadratic coefficient indicates that lipid content increases with FSF concentration up to the upper region of the experimental domain (10\u0026ndash;11.04% FSF; x₁ = +1 to +\u0026thinsp;1.41). This trend is consistent with runs 3 (10% FSF, 1% GSF), 4 (10% FSF, 5% GSF) and 6 (11.04% FSF, 3% GSF), which presented the highest lipid contents (9.70\u0026ndash;10.11%). In contrast, the non-significant or small coefficients for x₂ confirm that GSF did not markedly influence lipid content within the studied range (up to 5.83%).\u003c/p\u003e\n\u003cp\u003eProtein content followed a similar pattern and was modelled as:\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e(PROTEINS)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.20\u0026thinsp;+\u0026thinsp;0.29x\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.006x\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;0.05x\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.01x\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e \u0026minus;\u0026thinsp;0.01 x\u003csub\u003e1\u003c/sub\u003ex\u003csub\u003e2\u003c/sub\u003e (4)\u003c/p\u003e\n\u003cp\u003ewhere: x\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;FSF (%), x\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;GSF (%).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, increasing FSF concentration resulted in a proportional rise in protein content. Formulations containing 10\u0026ndash;11.04% FSF (x₁ = +1 to +\u0026thinsp;1.41) yielded the highest protein values, with runs 3, 4 and 6 reaching 16.41\u0026ndash;17.28%. GSF concentration did not significantly affect protein content within the evaluated range. Together, these models highlight FSF as the key driver of nutritional enhancement, while GSF plays a minor role in macronutrient enrichment but contributes structurally via its hydrated galactomannan matrix.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;2 shows the SEM micrographs of FSF, GSF, the control bread crumb, and the enriched bread crumb (Run 4: 10% FSF, 5% GSF). The microstructural contrasts among these samples help clarify how FSF and GSF interact within the bread matrix and contribute to their functional effects in the formulations. The micrograph of FSF shows irregular particles with angular fragments, compact aggregates and rough surfaces, typical of protein- and fiber-rich seed flours. This morphology reflects the presence of galactomannans, proteins and lipids adhering to the matrix and suggests high water-binding capacity and interaction potential with gluten, explaining the improved dough viscoelasticity in formulations with higher FSF levels [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe GSF micrograph reveals a compact, sponge-like and cohesive gel, characteristic of hydrocolloid-rich systems. Its smooth, continuous structure indicates a three-dimensional galactomannan network with strong water retention and gas-cell stabilization capacity, supporting dough hydration without impairing gluten development and consistent with its high moisture and aw values. The control bread crumb exhibited a dense, compact starch\u0026ndash;protein matrix with low porosity. Starch granules remained partially embedded in the protein phase, suggesting limited gelatinization and weaker structural integration. This morphology is consistent with a less stable gas-cell system and aligns with the lower specific volume, alveoli number and circularity observed [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn contrast, Run 4 (10% FSF, 5% GSF) showed a more cohesive, continuous crumb with fused starch domains, thinner cell walls and larger interconnected pores, indicating enhanced gelatinization and stronger interactions among FSF components (proteins, lipids, galactomannans) and gluten. This expanded, elastic microstructure explains the higher specific volume and improved alveoli characteristics [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Overall, the SEM results confirm that FSF reinforces the matrix through its protein, lipid and fiber fractions, whereas GSF contributes hydration and gas-cell stability. Together, they generate a more porous, homogeneous and mechanically stable crumb, consistent with the superior technological properties of the enriched breads.\u003c/p\u003e\n\u003cp\u003eThe CLSM image (Fig.\u0026nbsp;3) of FSF (A) shows a heterogeneous fluorescence pattern with red, blue and purple regions corresponding to proteins, polysaccharides and fiber-rich domains. This distribution reflects FSF\u0026rsquo;s nutritional density and its capacity for water binding and interaction with gluten, supporting the improved dough viscoelasticity in the enriched breads [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. The GSF micrograph (B) presents a cohesive, uniformly fluorescent matrix typical of hydrocolloid gels, indicating a well-organized galactomannan network. This structure explains its high-water retention and contribution to gas-cell stabilization without disrupting gluten development.\u003c/p\u003e\n\u003cp\u003eThe control crumb (C) displayed a dense, poorly organized matrix with weak fluorescence and limited protein\u0026ndash;starch definition, consistent with lower loaf volume and reduced aeration. This reflects the limited structural efficiency of the wheat matrix without added biomaterials [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast, the enriched bread (D) exhibited a more open and continuous structure with clearer protein\u0026ndash;starch interactions and more uniformly distributed fluorescent domains, supporting the improved macroscopic quality of the enriched breads. The confocal microscopy results confirm the synergistic action of FSF and GSF in breadmaking. FSF strengthens the matrix through its protein- and fiber-rich composition, whereas GSF enhances hydration and gas-cell stability. Together, they promote a more cohesive, elastic and aerated crumb, consistent with the improved physicochemical and technological attributes observed.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrated that galactomannan-rich ingredients obtained from \u003cem\u003eCaesalpinia pulcherrima\u003c/em\u003e seeds, applied as seed flour (FSF) and seed gel (GSF), act as functional biomaterials in wheat bread, simultaneously modulating technological quality, nutritional composition and microstructure. Within the ranges evaluated using a central composite design, the incorporation of FSF and GSF increased loaf specific volume, enhanced crumb porosity and circularity, and maintained expansion index values comparable to the control. These effects indicate improved gas retention and a more aerated, homogeneous crumb without impairing dough expansion capacity.\u003c/p\u003e \u003cp\u003eThe proximate composition results showed that FSF is the main contributor to nutritional enrichment, leading to higher protein, lipid, ash and, to a lesser extent, fiber contents in enriched breads compared with the control. GSF, in turn, had minor impact on macronutrient levels but played a relevant structural role by contributing to a hydrated galactomannan matrix. Moisture and water activity values of the breads remained within typical ranges for pan bread and complied with regulatory limits, while pH and titratable acidity values confirmed that FSF and GSF are compatible with standard yeast fermentation and do not compromise product safety.\u003c/p\u003e \u003cp\u003eMicrostructural analyses by SEM and CLSM provided mechanistic support for the macroscopic and compositional findings. FSF reinforced the gluten\u0026ndash;starch matrix through its protein-, lipid- and fiber-rich particles, whereas GSF contributed a cohesive hydrocolloid network with high water-binding and gas-cell stabilization capacity. Together, these ingredients generated a more continuous, cohesive and porous crumb structure, explaining the improved technological performance of the enriched breads and establishing clear structure\u0026ndash;function relationships for \u003cem\u003eC. pulcherrima\u003c/em\u003e seed biomaterials in bakery systems.\u003c/p\u003e \u003cp\u003eOverall, the results highlight \u003cem\u003eC. pulcherrima\u003c/em\u003e seeds as a promising and underutilized tropical resource for the development of nutritionally enhanced and structurally tailored breads using clean-label, plant-based ingredients. Future studies should address sensory acceptance, shelf-life behavior and process scaling, as well as the incorporation of FSF and GSF into other cereal-based products, to further explore their potential in sustainable and functional food design.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics and consent to participate\u003c/h2\u003e \u003cp\u003e The collection of plant material used in this study complied with applicable institutional, local, and national guidelines and legislation. Pods/seeds of \u003cem\u003eCaesalpinia pulcherrima\u003c/em\u003e (L.) Sw. (Fabaceae) were collected from cultivated ornamental trees located at different sites on the campus of the Federal University of Cear\u0026aacute; (UFC), Fortaleza, Cear\u0026aacute;, Brazil (Latitude: 3\u0026deg;44'38.6\"S; Longitude: 38\u0026deg;34'47.3\"W). The plant material was taxonomically identified by Prof. Dr. Maria Iracema Bezerra Loiola (Curator, Herbarium Prisco Bezerra \u0026ndash; EAC). Voucher specimen(s) were deposited in the Herbarium Prisco Bezerra (EAC), Federal University of Cear\u0026aacute;, Fortaleza, Cear\u0026aacute;, Brazil, under accession number EAC 67246. This study did not involve human participants or animals; therefore, ethics approval and consent to participate are not applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to publish\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Rafael Audino Zambelli. Methodology: Neilane Gomes da Rocha, Elenilson Godoy Alves Filho. Investigation: Poliana Brito de Sousa, Neilane Gomes da Rocha, Camila de Carvalho Chaves. Formal analysis: Poliana Brito de Sousa, Camila de Carvalho Chaves. Resources: Ana Karoline Nogueira Freitas. Supervision: Ana Karoline Nogueira Freitas, Rafael Audino Zambelli. Validation: Elenilson Godoy Alves Filho. Project administration: Rafael Audino Zambelli. Writing \u0026ndash; original draft: Poliana Brito de Sousa. Writing \u0026ndash; review and editing: Elenilson Godoy Alves Filho, Rafael Audino Zambelli.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank IFPI, IFMA and Embrapa Meio-Norte for support and access to laboratory facilities used in this work. We also acknowledge the Federal University of Cear\u0026aacute; and the Analytical Center-UFC/CT-INFRA/MCTI-SISNANO/Pro-Equipment CAPES, which contributed to the success of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eL.C.N.M. Bezerra, C.E.M. Silva, H.O. Nascimento, P.B.L. 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Food Sci. Food Saf. \u003cb\u003e22\u003c/b\u003e(3), 2081\u0026ndash;2111 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1541-4337.13141\u003c/span\u003e\u003cspan address=\"10.1111/1541-4337.13141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"functional bakery products, galactomannans, legume-based ingredients, tropical biodiversity valorization","lastPublishedDoi":"10.21203/rs.3.rs-8977729/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8977729/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGalactomannan-rich ingredients from tropical legumes are promising biomaterials to design bakery products with improved structure and nutritional quality. This study evaluated the structure\u0026ndash;function relationships of \u003cem\u003eCaesalpinia pulcherrima\u003c/em\u003e seed flour (FSF) and seed gel (GSF) in wheat bread. A central composite design assessed the effects of FSF (3.96\u0026ndash;11.04%) and GSF (0.17\u0026ndash;5.82%) on technological properties, proximate composition and microstructure. FSF and GSF increased loaf specific volume (from 2.16 to 3.87 mL g⁻\u0026sup1;), alveoli number and circularity, while maintaining expansion index values comparable to the control, indicating improved gas retention without impairing dough expansion. FSF-enriched breads showed higher protein (up to 17.28%), lipid (up to 10.11%), ash and fiber contents than the control, confirming FSF as the main contributor to nutritional enrichment, whereas GSF mainly affected hydration and crumb structure. pH, moisture and water activity remained within typical ranges for pan bread and compatible with standard yeast fermentation. SEM and CLSM images revealed a more continuous and cohesive gluten\u0026ndash;starch matrix with thinner cell walls and more uniform gas cells in enriched crumbs, aligning with the improved technological performance. Overall, FSF and GSF act as functional biomaterials that valorize an underutilized tropical legume and support the development of nutritionally enhanced, structurally tailored wheat breads.\u003c/p\u003e","manuscriptTitle":"Galactomannan-rich ingredients from Caesalpinia pulcherrima seeds reorganize wheat bread microstructure and enhance technological and nutritional quality","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 17:35:59","doi":"10.21203/rs.3.rs-8977729/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-09T16:34:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-08T06:12:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159174582022540866834894225793234669864","date":"2026-03-04T03:53:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T19:00:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11558368246273269361272598934856911692","date":"2026-03-03T18:53:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-03T18:48:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-02T11:55:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-02T11:55:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Biophysics","date":"2026-02-26T12:08:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7f36a6dd-3fec-46ee-ace0-b6f88e5c9ff3","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T14:55:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 17:35:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8977729","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8977729","identity":"rs-8977729","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}