Structural and functional evolution of flaxseed protein during probiotic fermentation: Insights into proteolytic degradation, conformational rearrangement, and thermal property

Structural and functional evolution of flaxseed protein during probiotic fermentation: Insights into proteolytic degradation, conformational rearrangement, and thermal property | 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 Structural and functional evolution of flaxseed protein during probiotic fermentation: Insights into proteolytic degradation, conformational rearrangement, and thermal property Cong Min, Yukun Wang, Wenqian Gong, Xiangming Ye, Wen Su This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8463814/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Flaxseed protein (FP) is a promising plant-based protein source, yet its application in food systems is limited by its inherent structural characteristics. This study systematically investigated the structural and functional evolution of flaxseed protein (FP) during fermentation by Lactobacillus bulgaricus and Bifidobacterium lactis . The fermentation process induced significant time-dependent changes in particle size distribution, shifting from large aggregates (> 100 µm) to a homogenized system dominated by 10–100 µm particles, accompanied by the complete disruption of the native dense microstructure into sub‑50 µm dispersed particles. SDS‑PAGE analysis revealed progressive degradation of high‑molecular‑weight fractions (> 100 kDa) and accumulation of low‑molecular‑weight peptides (15–35 kDa), confirming extensive proteolysis. UV‑spectroscopy indicated a decrease in absorbance at 280 nm and a red‑shift in peak position, suggesting the release of aromatic amino acids and a transition toward disordered conformations. Differential scanning calorimetry demonstrated a marked reduction in denaturation temperature (from 149.27°C to 101.77°C) and an increase in enthalpy change (from 16.77 J/g to 22.80 J/g), reflecting decreased thermal stability and enhanced hydration potential. The results collectively delineate a three‑stage mechanism: initial protease‑driven hydrolysis (0–4 h), intermediate hydrophobic‑electrostatic recombination (4–8 h), and final formation of uniform particles (8–10 h). These structural modifications render fermented FP more suitable for gel‑based food applications by lowering its thermal transition requirements and improving water interaction. This work provides a theoretical foundation for the targeted fermentation design of plant proteins to tailor their techno‑functional properties. Flaxseed protein Probiotic fermentation Thermodynamic properties Structural reorganization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Flaxseed protein (FP), recognized as a high-quality plant-based protein, has garnered significant attention in the realm of health foods due to its substantial nutritional value and functional properties [ 1 , 2 ]. It is abundant in amino acids, encompassing 8 essential amino acids that constitute over 30% of its composition, and shares a comparable amino acid profile and nutritional attributes with soy protein. FP is particularly rich in aspartic acid, glutamic acid, and arginine; moreover, the content of seven essential amino acids, including phenylalanine, isoleucine, lysine, leucine, threonine, valine, and tryptophan, exceeds the adult recommended intake by the World Health Organization [ 3 , 4 ]. Compared to soy protein, FP boasts a higher arginine content and a lower lysine/arginine ratio, which positively influences muscle and immune function as well as cardiovascular health in the elderly, particularly those with swallowing difficulties. Beyond its role as a nutritional supplement, FP also holds the potential to enhance the functional properties of food [ 5 ]. It can serve as a texture regulator to enhance the hardness, elasticity, color, appearance, and flavor of baked goods or pastries such as bread, pizza, and biscuits. Additionally, FP can improve the viscoelasticity and water retention of snacks or cereal products, impart a unique hardness and viscosity to noodle products like pasta, and decrease cooking losses in meat products such as sausages [ 6 ]. Nevertheless, natural FP faces limitations such as poor solubility, low thermal stability, and limited functionality, which severely impede its application in high-value products [ 7 , 8 ]. In recent years, microbial fermentation modification has emerged as a strategy for enhancing plant protein quality, owing to its environmentally friendly and efficient nature [ 9 , 10 ]. Previous research has demonstrated that microbial fermentation methods, including those using Lactobacillus, Lactobacillus rhamnosus, and Lactobacillus plantarum, can effectively eliminate toxic components such as cyanogenic glycosides from flaxseed. These methods also influence the formation of biogenic amines and volatile compounds in flaxseed [ 11 ]. For functional proteins, employing microbial strains like Lactobacillus plantarum, Bacillus subtilis, and Aspergillus oryzae for fermentation can break down protein molecules, resulting in a large number of small molecule peptides. Many of these peptides possess various physiological activities, such as inhibiting angiotensin-converting enzyme, antioxidant properties, antibacterial effects, and anti-diabetic potential, indicating their potential as bioactive peptides [ 12 – 14 ]. Moreover, the digestibility and thermal stability of FP fermented with probiotics can be regulated [ 15 , 16 ]. Consequently, microbial fermentation holds significant promise for improving the functional properties of FP and equivalent plant proteins, as well as their application in the functional foods domain. However, research on its specific mechanisms of action and processes of change remains limited. In this study, we innovatively used probiotic complexes (Lactobacillus bulgaricus: Bifidobacterium lactis = 1:2) for fermentation modification of FP. Based on preliminary experiments, it was found that the two bacteria had a synergistic effect under this ratio, which could more efficiently hydrolyze FP macromolecular proteins, and the degree of thermal stability reduction was significantly better than that of single strain fermentation. The structural evolution of fermented flaxseed protein (FFP) during the first 10 h of fermentation was systematically investigated using laser particle size analysis, scanning electron microscopy (SEM), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet spectroscopy, and differential scanning calorimetry (DSC). This research provides a scientific foundation for the precise regulation of fermentation processes and promotes the efficient utilization of plant protein resources by establishing a multidimensional correlation map of structure, thermal performance, and function. 2. Materials and methods 2.1. Materials and chemical reagent FP (≥ 81% protein, 3.62% moisture, 5.24% lipids) was obtained from Shaanxi Kangduopu Biotechnology Co., Ltd. (Xi'an, Shaanxi, China). Lactobacillus bulgaricus and Bifidobacterium lactis (both with a viable count of 210¹⁰ cfu) were sourced from Shaanxi Benhe Bioengineering Co., Ltd. (Xi'an, Shaanxi, China). Water is deionized. All other reagents were analytical grade reagents. 2.2. Fermentation of flaxseed protein Weigh 30.0 g of FP powder add deionized water to prepare a suspension. Stir the suspension magnetically at room temperature for 10 minutes, then dilute it to 100 mL to achieve a final concentration of 30% FP suspension. The FP suspension was sterilized in a 95 ℃ water bath for 10 minutes and subsequently cooled rapidly to 37 ℃. Following the method of Xu et al. [ 17 ], probiotic fermentation agents (1% Lactobacillus bulgaricus and 2% Bifidobacterium lactis , both with a viable cell count of 2×10¹⁰ cfu) were added to the FP suspension and fermented at 37°C for 2, 4, 6, 8, and 10 h for degradation. The unfermented natural FP (0 h) served as the control group. Fermentation - degradation FP samples with different fermentation times were frozen overnight at -80 ℃, followed by freeze - drying at -80 ℃ for 48 h. The dried samples were then ground into powder for subsequent use. 2.3. Particle size determination Freeze-dried FFP powder samples with varying fermentation times were added to deionized water to prepare a solution with a concentration of 4 mg/mL. Magnetic stirring was performed for 2 h to ensure complete dissolution of the protein and uniform dispersion of the particles. The size of the protein particles was measured using a laser nanoparticle size analyzer. The parameters were as follows: the dispersant was a 10 mmol/L phosphate buffer solution (pH 7.0), and the dispersant refractive index was 1.3318. The measurement was repeated three times. 2.4. Scanning electron microscopy The method described by Min et al. [ 18 ] was followed with minor modifications. fine adjustments were made by fixing freeze-dried FFP powder samples with varying fermentation times on a sample stage and performing a gold spraying treatment on the samples. Subsequently, observation and photography were conducted using a scanning electron microscope at a voltage of 20 kV. 2.5. Protein electrophoresis detection Dissolve freeze-dried powder samples of FFP with varying fermentation times in deionized water, stir magnetically at room temperature for 10 minutes, filter through a 0.45 µm filter, and prepare an FFP solution with a concentration of 1 mg/mL. Add an appropriate amount of SDS-PAGE loading buffer, mix well, boil at 95 ℃ for 5 minutes, and immediately place on ice; centrifuge at 12,000 rpm at 4 ℃ for 10 minutes. Then, take 20 µL of each supernatant for electrophoresis separation (with a concentrated gel concentration of 4% and a separated gel concentration of 15%), applying a constant voltage of 80 V in the concentrated gel area and 120 V in the separated gel area. 2.6. UV full wavelength scanning detection Prepare a solution with a mass concentration of 2.0 mg/mL using FFP freeze-dried powder for testing. Transfer approximately 3 mL to a quartz colorimetric dish and use a UV-visible spectrophotometer to perform a full wavelength UV scan. Set the scanning wavelength range from 200 to 600 nm, with a slit width of 1.0 nm and a sampling interval of 0.5 nm, and conduct the scan at a slow speed. 2.7. Thermal characteristic analysis Take 3.0 mg samples of freeze-dried FFP powder with varying fermentation times, seal them in an aluminum crucible, and analyze using a differential scanning calorimeter. Set the instrument parameters as follows: a heating rate of 10.0 ℃/min, within a temperature range of 80 ℃ to 180 ℃. Record the detection values: starting temperature (To), peak temperature (Tp), final temperature (Tc), and enthalpy value (ΔH). 2.8. Data analysis All tests were conducted in triplicate, and the data are expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA) and Duncan's test were used to perform the statistical analysis with SPSS 22 software (IBM, NY, USA). A P-value of less than 0.05 was considered statistically significant. 3. Results and discussion 3.1 Particle size analysis of fermented flaxseed protein The laser particle size analysis results indicated that the particle size distribution of FFP during the fermentation process exhibited significant time-dependent evolution characteristics (Table 1 ). At the unfermented stage (0 h), the sample particle size distribution was dominated by large particles (≥ 100 µm, accounting for 25.71%) and medium particles (10–100 µm, accounting for 44.02%). As the fermentation process progressed (2–10 h), the proportion of particles with a diameter greater than 100 µm sharply decreased to 3.20% (p < 0.01), while the proportion of particles with a diameter between 10–100 µm significantly increased to 64.1%. It is noteworthy that the proportion of < 0.1 µm ultrafine particles decreased from 5.96% to 2.38%, suggesting that degradation products may migrate to larger particle size ranges through dissolution or recombination mechanisms [ 19 – 21 ]. The dynamic evolution of particle size homogenization distribution during fermentation reveals a two-stage mechanism of action [ 22 , 23 ]. On one hand, the protease-dominated primary degradation stage (0–4 h) rapidly reduces large particles (100 µm). This process originates from the specific hydrolysis of peptide bonds by endonucleases secreted by probiotic fermentation agents (Lactobacillus bulgaricus and Bifidobacterium lactis) [ 24 , 25 ]. On the other hand, there is a secondary aggregation stage of degradation products (6–10 h), where the proportion of medium particles (10–100 µm) continues to increase. The possible pathway for its formation is the formation of hydrophobic core aggregates and charged groups through electrostatic interactions after the exposure of hydrophobic peptide segments to construct a stable network structure [ 26 , 27 ]. Table 1 Particle size of the FFP following various fermentations. Means with different letters (a-f) differ significantly (P < 0.05) among the data in the same column Sample 100 µm 0 h 5.96 ± 0.83 a 8.39 ± 0.96 b 15.92 ± 0.36 d 44.02 ± 1.15 e 25.71 ± 1.62 a 2 h 4.02 ± 0.51 b 9.96 ± 0.62 ab 16.92 ± 0.29 c 52.11 ± 1.73 d 16.99 ± 0.85 b 4 h 3.67 ± 0.26 bc 10.05 ± 0.89 ab 17.20 ± 0.36 bc 56.80 ± 1.26 c 12.28 ± 0.91 c 6 h 3.12 ± 0.19 c 10.57 ± 0.91 a 17.56 ± 0.25 b 59.61 ± 1.19 b 9.14 ± 0.73 d 8 h 3.06 ± 0.21 cd 11.06 ± 0.85 a 18.13 ± 0.29 b 62.56 ± 1.27 a 5.19 ± 0.29 e 10 h 2.38 ± 0.17 d 11.16 ± 0.79 a 19.16 ± 0.23 a 64.10 ± 1.25 a 3.20 ± 0.52 f 3.2. Microstructure of fermented flaxseed protein During the fermentation process, the appearance of FFP powder exhibits regular changes over time (as shown in Fig. 1 ). In the early stage of fermentation (0 h), the powder has a lighter color, uneven texture, and a tight structure with obvious clumping. As the fermentation time is extended to 2–4 h, the color of the powder gradually deepens, the texture begins to become loose, and the phenomenon of clumping gradually decreases. In the middle stage of fermentation (6–8 h), the color further turns brown due to Maillard reaction or accumulation of fermentation products, appearing light brown, with a significantly loose texture, and the particle size decreases and gradually becomes uniform. In the late stage of fermentation (10 h), the powder color is the darkest, close to burnt yellow or brown, and the particle size gradually becomes uniform, consistent with the data of particle size. The overall trend indicates that the longer the fermentation time, the darker the powder color, the looser the texture, and the gradually improved structural uniformity. Upon SEM observation, it was revealed that the microstructure of FFP undergoes significant stage changes throughout the fermentation process (Fig. 1 ). Prior to fermentation (at 0 h), FFP displays a dense, block-like or layered structure with a smooth surface, suggesting that its native conformation remains intact and intermolecular forces, (such as hydrogen bonding and hydrophobic interactions, are stable. In the initial stage of fermentation (2–4 h), the sample's particle size ranges from 50–100 µm, indicating that the probiotic fermentation agents (Lactobacillus bulgaricus and Bifidobacterium lactis) have begun to act on peptide bonds, resulting in localized structural depolymerization. During the intermediate stage of fermentation (6–8 h), FFP becomes further loosened, potentially due to intermolecular cross-linking caused by the exposure of hydrophobic groups, (uch as phenylalanine and leucine residues. In the late stage of fermentation (10 h), the microstructure is completely fragmented, presenting dispersed particles with a size of less than 50 µm [ 28 , 29 ]. The findings indicate that fermentation transforms FFP from a dense block structure into nano-sized dispersed particles, a process dominated by the degradation of probiotic fermentation agents and achieving function-oriented recombination through hydrophobic electrostatic equilibrium. As fermentation time progresses, the particle size of FFP powder becomes increasingly uniformly distributed [ 30 ]. This process is closely linked to the degradation of proteins, hydrophobic interactions, and structural stability changes of probiotic fermentation agents, offering a structural basis for their functional modification (such as active peptide release and processing performance optimization). 3.3. The effect of fermentation degradation on the relative molecular weight of flaxseed protein The molecular weight distribution of peptide segments in fermentation products is another important indicator reflecting the extent of protein fermentation [ 31 , 32 ]. This article analyzed the molecular weight distribution of peptide segments in FFP after degradation at different times using the SDS-PAGE method, and the results are shown in Fig. 2 . During fermentation, it was revealed that unfermented FFP (0 h) exhibited significant bands in the molecular weight > 100 kDa region, indicating its natural state as high molecular weight aggregates or complexes. As the fermentation time extends (2–10 h), the intensity of the bands greater than 100 kDa diminishes progressively, while there is a corresponding rise in the intensity of bands within the 15–35 kDa low molecular weight range. The degradation process of FFP exhibits marked time-dependent characteristics [ 33 , 34 ]. In the early stage of fermentation (0–4 h), rapid degradation occurs, and the content of > 100 kDa protein decreases within 4 h, indicating that the probiotic fermentation agent is rapidly activated in the early stage of fermentation, preferentially attacking the loose structural domains of FFP (such as the α-helix region). There is a dynamic equilibrium during the mid-fermentation stage (4–8 h), during which the degradation rate slows down, possibly due to the remaining substrate being a resistant structure (β-folding cluster or disulfide bond stable region), with hydrophobic core exposure driving aggregation and forming stable secondary structures [ 35 , 36 ]. In the final stage of fermentation, most of the proteins are depolymerized into small peptides, maximizing their functional activity. Unfermented FP exists in the form of oligomers (180 kDa), while smaller fragments of 10–15 kDa gradually increase with fermentation time after fermentation, indicating that the dissociation of non-covalent bonds (hydrophobic interactions, etc.) dominates subunit separation [ 37 , 38 ]. The SDS-PAGE experimental results further confirmed the observation results of particle size analysis and scanning electron microscopy, indicating that with the prolongation of fermentation time, the structure of FFP undergoes further dissociation, gradually forming more uniform and smaller particles. This study provides a theoretical basis for the high-value utilization of FP and lays a technical foundation for precise regulation of fermentation processes. 3.4 UV absorption spectrum of fermented flaxseed protein The FFP sample was scanned across the full wavelength range from 200 nm to 600 nm using a UV spectrophotometer, and its UV absorption spectrum is depicted in Fig. 3 . In the UV absorption spectrum, the absorption peak near 280 nm is primarily attributed to aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). The absorbance at this wavelength in the spectrum markedly decreases with fermentation time (0 h − 10 h), suggesting that protein degradation may be occurring, resulting in the release of aromatic amino acids or structural damage. The overall shape of the absorbance curve, including peak width and peak position, shifts at various fermentation time points, which may reflect changes in the conformation of protein secondary structures, (such as alpha helices and beta sheets [ 39 ]. During the late stage of fermentation (6–10 h), the absorbance stabilized compared to the control group (0 h), suggesting that the protein structure might have reached a dynamic equilibrium or that microbial metabolic activity had slowed. UV-visible spectroscopy analysis reveals that the structural characteristics of FFP undergo significant dynamic evolution throughout the fermentation process. The absorbance at 280 nm decreases as fermentation time (extends from 0 to 10 h, indicating the release or structural damage of aromatic amino acids (tryptophan, tyrosine) due to protein degradation. This is consistent with the gradual disappearance of high molecular weight bands in SDS-PAGE. Additionally, the spectral peak position in the 250–300 nm range shifts from 275 nm to 282 nm, suggesting a transition from an ordered to a disordered protein secondary structure, potentially driven by molecular rearrangement induced by the exposure of hydrophobic groups. [ 40 ]. The UV absorption spectroscopy results indicate that the fermentation modification of FFP involves protease-mediated degradation and conformational relaxation. 3.5 Thermal characteristics of FFP fermented at different times ​The thermal stability of proteins is an important factor affecting the processing quality of gel food produced through heat treatment [ 41 – 43 ]. To comprehensively evaluate the influence and function of probiotic fermentation on the thermal characteristics of proteins, this study conducted DSC analysis on FFP at different fermentation stages. The DSC thermograms revealed significant changes in the thermal characteristics of FFP throughout the fermentation process (Fig. 4 ). The fermentation of probiotics markedly diminishes the thermal stability of FFP, evidenced by a reduction in the denaturation temperature and an increase in structural heterogeneity. This phenomenon is closely associated with protein degradation, the destruction of the secondary structure, and the exposure of hydrophobic groups [ 44 – 45 ]. The unfermented sample (0 h) displayed a single endothermic peak at 149.27 ± 8.22°C, indicating that natural FP has high thermal stability and an ordered secondary structure. Its tightly folded secondary structures, comprising alpha helices and beta folds, require high energy for melting. As fermentation progresses (2–10 h), the primary endothermic peak gradually shifts to lower temperatures (10 h: 101.77 ± 8.69°C, p < 0.01), and a decrease in denaturation temperature suggests the disruption of stable interactions, (such as hydrogen bonding and hydrophobic stacking. During the fermentation process, which lasts 2 to 10 h, the peak position shifts towards lower temperatures. This shift indicates a loosening of the protein structure, potentially leading to a weakening of intramolecular forces, such as hydrogen bonding and hydrophobic interactions (Dickson et al., 2025). According to Table 2 , as fermentation progresses, the starting temperature (To) of FFP decreases from 132.75°C (at 0 h) to 101.77°C (at 10 h), a decrease of 23.3%, indicating a reduction in the energy required for fermentation to disrupt the protein crystal structure. The peak temperature (Tp) decreases from 149.27°C (at 0 h) to 115.43°C (at 10 h), a decrease of 22.7%, reflecting a reduction in the orderliness and thermal stability of FFP molecules. The termination temperature (Tc) decreases from 170.51°C (at 0 h) to 129.99°C (at 10 h), a decrease of 23.8%, indicating that the gelatinization process is accelerated and the efficiency of molecular chain disentanglement is improved. The enthalpy change (ΔH) continues to increase, rising from 16.77 J/g at 0 h to 22.80 J/g at 10 h—an increase of 36%. This suggests that the gelatinization process requires more heat absorption. It indicates that the crystal area is damaged or the disordered structure has increased, which in turn suggests that its water absorption is enhanced. The results of DSC and UV detection of peak displacement and structural disorder confirmed each other, indicating that fermentation modified FFP is more suitable for gel food. Table 2 Onset temperature (To), peak temperature (Tp), concluding temperature (Tc), and enthalpy (ΔH) of the FFP after various fermentation. Means with different letters (a-e) differ significantly (P < 0.05) among the data in the same column. Sample T O (℃) T P (℃) T C (℃) ΔH (J/g) 0 h 132.75 ± 3.52 a 149.27 ± 3.22 a 170.51 ± 3.61 a 16.77 ± 0.26 e 2 h 121.93 ± 2.26 b 139.86 ± 2.63 b 163.05 ± 2.86 b 18.14 ± 0.76 d 4 h 111.03 ± 2.14 c 129.82 ± 1.69 c 148.47 ± 2.22 c 19.76 ± 0.22 c 6 h 107.33 ± 1.37 cd 122.06 ± 3.03 d 141.24 ± 1.25 d 20.22 ± 0.28 c 8 h 104.87 ± 1.58 d 118.13 ± 2.26 d 132.32 ± 2.91 e 21.05 ± 0.32 b 10 h 101.77 ± 2.69 d 115.43 ± 2.61 e 129.99 ± 1.89 e 22.80 ± 0.26 a 3.6 Schematic mechanism Under the influence of probiotic fermentation, where L. bulgaricus and B. lactis maintain a symbiotic relationship at a ratio of 1:2, the structure and molecular forces of FP undergo significant changes, particularly during the critical transition period of 8–10 h. Based on the molecular weight and thermal characteristics of flaxseed protein (FP) fermented over various periods, this study proposes a mechanism logic model of "microbial protease hydrolysis, structure recombination, and performance alteration" (as depicted in Fig. 5 ). The study achieved a probiotic-driven structural reconstruction of FP through the regulation of fermentation using Lactobacillus bulgaricus and Bifidobacterium lactis. Time characterization revealed a three-stage transformation mechanism. Within 0–4 h, microbial proteases preferentially hydrolyze hydrophobic peptide bonds, thereby reducing large molecular aggregates (> 100 µm; p 100 kDa), as indicated by particle size distribution and SDS-PAGE analysis. Subsequently, hydrophobic electrostatic recombination (4–8 h) formed a metastable porous network. The red shift of the UV absorption peak at A280 indicates that the secondary structure is disordered. The final fermentation stage, which lasted 8–10 h, resulted in uniform sub-50 µm particles. Additionally, the DSC results indicated that the gel transition temperature decreased by 23.3–23.8%, and the ΔH increased by 36%, signifying a significant alteration in its thermal behavior. This protease-mediated cascade reaction, which converts ordered aggregates into disordered particle systems, provides a feasible strategy to reduce the restrictions of FP in the food preparation process due to high deformation temperatures. 4. Conclusions This study examined the dynamic regulatory mechanisms of probiotic fermentation (Lactobacillus bulgaricus and Bifidobacterium lactis) on the structure and thermal properties of FP using a multi-scale characterization system, and established a multidimensional evaluation system covering particle size, molecular weight, thermal characteristics, and conformation. The results showed that the particle size of FP evolved in a time-dependent manner: within 0-4 h of fermentation, protease specific hydrolysis of peptide bonds led to a sharp decrease in the proportion of particles larger than 100 μ m, while the proportion of particles larger than 10-100 μ m increased. SDS-PAGE results showed the degradation of polymer aggregates (>100 kDa) accompanied by the release of aromatic amino acids; Fermentation for 4-8 h enters the hydrophobic driven recombination stage, forming a stable network through hydrophobic electrostatic equilibrium; At 10 h, FP completely dissociated into dispersed particles<50 μ m (SEM verification). In terms of structure, the UV spectrum shows a decrease in absorbance and a red shift at 280 nm, indicating a decrease in the orderliness of the secondary structure; The DSC results further indicate that fermentation reduces the thermal denaturation temperature (To, Tp, Tc) and increases ΔH, indicating that hydrogen bonding and hydrophobic interactions are disrupted, leading to crystal structure collapse and increased disorder, requiring gelatinization to absorb more heat. This study clarifies that probiotic fermentation destroys the ordered structure of FP through enzymolysis, reduces its thermal stability and improves its heat absorption capacity. This feature is highly consistent with the process requirements of plant based meat products for low heat treatment and high gelatinization, which is expected to improve the caking and texture problems of traditional FP in thermal processing. In the future, we can focus on optimizing the application ratio of fermented FP in plant-based meat, and combine molecular simulation to deeply analyze the structure-activity relationship of key peptide segments, in order to achieve precise regulation and industrial application of fermentation technology. Declarations Declaration of competing interest The authors declare no conflict of interest regarding the publication of this paper, and the manuscript is approved by all authors for publication. Funding The financial support from the Scientific Research Program Funded by Education Department of Shaanxi Provincial Government (Program No. 24JK0371), the Scientific Research Project for Talented Scholars of Shaanxi University of Technology (SLGRC202407, SLGRCQD2203), National Natural Science Foundation of China Cultivation Project (SLGGZRPY14). Author Contribaution Cong Min : Conceptualization, Methodology, Formal analysis, Funding acquisition, Resources, Supervision, Writing – original draft. Yukun Wang : Methodology, Investigation, Software, Formal analysis, Data curation. Wenqian Gong: Methodology, Resources. Xiangming Ye : Formal analysis, Methodology. Wen Su : Conceptualization, Resources, Supervision, Writing - review & editing. Data Availability Data availability： Data will be made available on request. References Wang B, Han J, Liu C, Zhang J, Qi Y (2025) Flaxseed protein content prediction based on hyperspectral wavelength selection with fractional order ant colony optimization. Front Nutr 12:1551029. https://doi.org/10.3389/fnut.2025.1551029 Ren J, Fu J, Zhao X (2024) Structural characterization and performance analysis of flaxseed isolate protein. Trans Chin Soc Agricultural Eng 40(5):326–336. https://doi.org/10.11975/j.issn.1002-6819.202312060 Jaroová M, Roudnická P, Bárta J, Zdráhal Z, Bártová V, Stupková A, Lorenc F, Bjelková M, Kyselka J, Jaroová E (2024) Proteomic profile of flaxseed (Linum usitatissimum L.) products as influenced by protein concentration method and cultivar. Foods 13(9):1288. https://doi.org/10.3390/foods13091288 Kajla P, Sharma A, Sood DR (2015) Flaxseed—a potential functional food source. J Food Sci Technol 52(4):1857–1871. https://doi.org/10.1007/s13197-014-1293-y Sharma M, Saini CS (2022) Amino acid composition, nutritional profiling, mineral content and physicochemical properties of protein isolate from flaxseeds (Linum usitatissimum). J Food Meas Charact 16(1):38–48. https://doi.org/10.1007/s11694-021-01221-0 Rabetafika HN, Van Remoortel V, Danthine S, Paquot M, Blecker C (2011) Flaxseed proteins: Food uses and health benefits. Int J Food Sci Technol 46(2):221–228. https://doi.org/10.1111/j.1365-2621.2010.02477.x Imran S, Munir S, Altemimi AB, Fatima I, Rabail R, Batool I (2024) Therapeutic implications of flaxseed peptides and bioactive components against various diseases. J Funct Foods 119:106324. https://doi.org/10.1016/j.jff.2024.106324 Kaushik P, Dowling K, McKnight S, Barrow CJ, Wang B, Adhikari B (2016) Preparation, characterization and functional properties of flax seed protein isolate. Food Chem 197:212–220. https://doi.org/10.1016/j.foodchem.2015.09.106 D'Almeida AP, Neta AAI, de Andrade Lima MATL (2024) Plant-based probiotic foods: Current state and future trends. Food Sci Biotechnol 33(15):3401–3422. https://doi.org/10.1007/s10068-024-01533-z Zhou Y, Xu Y, Song S, Zhan S, Li X, Wang H, Zhu Z, Yan L, Peng Y, Xie C (2024) Effect of different probiotic fermentations on the quality of plant-based hempseed fermented milk. Foods 13(24):4076. https://doi.org/10.3390/foods13244076 Bartkiene E, Schleining G, Juodeikiene G, Vidmantiene D, Krungleviciute V (2014) The influence of lactic acid fermentation on biogenic amines and volatile compounds formation in flaxseed and the effect of flaxseed sourdough on the quality of wheat bread. LWT-Food Sci Technol 56(2):445–450. https://doi.org/10.1016/j.lwt.2013.12.006 Wu M, Wang L, Li D, Wang Y, Mao ZH, Chen XD (2012) The digestibility and thermal properties of fermented flaxseed protein. International Journal of Food Engineering 8(4): Article 23. https://doi.org/10.1515/1556-3758.2178 Wang Z, Liu X, Li S, Fang Y, Cao Y (2025) Formation and morphology of flaxseed protein isolate amyloid fibrils as governed by NaCl concentration. Food Hydrocolloids 166:111300. https://doi.org/10.1016/j.foodhyd.2025.111300 Albe-Slabi S, Mesieres O, Beaubier S, Beau L, Aymes A, Roques-Carmes T, Sadtler V, Kapel R (2025) The role of extraction and purification conditions in flaxseed protein isolate production and its emulsifying properties. LWT-Food Sci Technol 215:117170. https://doi.org/10.1016/j.lwt.2024.117170 Pihlanto A, Johansson T, Mäkinen S (2012) Inhibition of angiotensin I-converting enzyme and lipid peroxidation by fermented rapeseed and flaxseed meal. Eng Life Sci 12(4):450–456. https://doi.org/10.1002/elsc.201100137 Wu M, Wang L, Li D, Wang Y, Mao ZH, Chen XD (2012) The digestibility and thermal properties of fermented flaxseed protein. International Journal of Food Engineering 8(4): Article 23. https://doi.org/10.1515/1556-3758.2178 Xu X, Cui H, Xu J, Yuan Z, Liu X, Fan X, Li J, Zhu D, Liu H (2022) Effects of different probiotic fermentations on the quality, soy isoflavone and equol content of soy protein yogurt made from soy whey and soy embryo powder. LWT-Food Sci Technol 157:113096. https://doi.org/10.1016/j.lwt.2022.113096 Min C, Wang Y, Li Y, Zhu Z, Li M, Chen W, Yi J, Liu M, Feng L, Cao Y (2025) Effects of transglutaminase on the gelation properties and digestibility of pea protein isolate with resonance acoustic mixing pretreatment. Food Chem 469:142534. https://doi.org/10.1016/j.foodchem.2024.142534 Chen Y, Peng C, Chen X, Imran M, Zhang H, Sakandar HA (2023) Impact of fermentation on antinutritional factors and protein degradation of legume seeds: A review. Food Reviews Int 39(3):1227–1249. https://doi.org/10.1080/87559129.2021.1931300 Toure M, Crews CM (2016) Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew Chem Int Ed 55(6):1966–1973. https://doi.org/10.1002/anie.201507978 Pei Y, Hinchliffe BA, Minelli C (2021) Measurement of the size distribution of multimodal colloidal systems by laser diffraction. ACS Omega 6(22):14049–14058. https://doi.org/10.1021/acsomega.1c00411 Li P, Xie C, Zeng Q, Yuan Y (2023) Effect of different hydrophobic soybean isolated peptides and their zinc complexes on the growth and fermentation of Lactobacillus bulgaricus. Int J Food Sci Technol 58(12):6411–6420. https://doi.org/10.1111/ijfs.16752 Liu Y, Fei Y, Li C, Cheng J, Xue F (2024) Impact of probiotic fermentation on the physicochemical properties of hemp seed protein gels. Polymers 16(21):3032. https://doi.org/10.3390/polym16213032 Kumari K, Kashyap P, Chakrabarti P (2023) Germination and probiotic fermentation: A way to enhance nutritional and biochemical properties of cereals and millets. Food Sci Biotechnol 33(3):505–518. https://doi.org/10.1007/s10068-023-01401-2 Oliveira RPS, Florence ACR, Perego P, Oliveira MN, Converti A (2011) Use of lactulose as prebiotic and its influence on the growth, acidification profile and viable counts of different probiotics in fermented skim milk. Int J Food Microbiol 148(2):111–117. https://doi.org/10.1016/j.ijfoodmicro.2010.11.011 Lawson EQ, Sadler AJ, Harmatz D, Brandau DT, Micanovic R, MacElroy RD, Middaugh CR (1981) A simple experimental model for hydrophobic interactions in proteins. Biochemistry 20(14):3995–4000. https://doi.org/10.1038/npg.els.0002975 Fragneto G, Su TJ, Lu JR, Thomas RK, Rennie AR (2000) Adsorption of proteins from aqueous solutions on hydrophobic surfaces studied by neutron reflection. Phys Chem Chem Phys 2(22):5214–5221. https://doi.org/10.1039/B004221H Fang X, Ye H, Chen MD, Chen XD, Chen MH, Chen JY, Kong WH, Wang QQ, Zhang ZY (2024) Effects of co-fermentation of lactobacillus plantarum and inulin on beany flavor and physicochemical quality of soy yogurt in a multiple lactic acid bacteria fermentation system. Cereal Chem 101(1):248–262. https://doi.org/10.1002/cche.10741 Tindjau R, Chua J-Y, Liu SQ (2023) Growth, substrate, and metabolite changes of probiotic Bifidobacterium animalis subsp. lactis in soy (tofu) whey. Fermentation 9(12):1024. https://doi.org/10.3390/fermentation9121024 Fan Q, Liu L, Wang L, Yang R, Liu X, Dong Y, Zeng X, Liu X, Du Q, Wu Z, Pan D (2025) Nanocoating of quinoa protein and hyaluronic acid enhances viability and stability of Limosilactobacillus fermentum RC4 microcapsules. Int J Biol Macromol 307(Pt 1):141863. https://doi.org/10.1016/j.ijbiomac.2025.141863 Kravchenko IV, Furalyov VA, Pshennikova ES, Fedorov AN, Popov VO (2024) The effect of fermentation by lactobacilli on the functional–technological properties of pea protein isolates. Appl Biochem Microbiol 60(6):1388–1397. https://doi.org/10.1134/S0003683824605493 Ross PD, Rekharsky MV (1996) Thermodynamics of hydrogen bond and hydrophobic interactions in cyclodextrin complexes. Biophys J 70(3):1231–1239. https://doi.org/10.1016/S0006-3495(96)79415-8 Arai T, Mio K, Onoda H, Chavas LMG, Umena Y, Sasaki YC (2023) The blinking of small-angle X-ray scattering reveals the degradation process of protein crystals at microsecond timescale. Int J Mol Sci 24(23):16640. https://doi.org/10.3390/ijms242316640 Wei G, Regenstein JM, Zhou P (2021) The fermentation-time dependent proteolysis profile and peptidomic analysis of fermented soybean curd. J Food Sci 86(11):4182–4192. https://doi.org/10.1111/1750-3841.15823 Ding Y, Yang Q, Wang M, He W, Dai W, Tang X (2024) Flavor and nutritional characteristics of surimi product with lactiplantibacillus plantarum as a reinforcing starter culture. Food Bioscience 62:105229. https://doi.org/10.1016/j.fbio.2024.105229 Zotta T, Piraino P, Ricciardi AM, McSweeney PLH, Parente E (2006) Proteolysis in model sourdough fermentations. J Agric Food Chem 54(11):4078–4085. https://doi.org/10.1021/jf052504s Ibarra-Molero B, Naganathan AN, Sanchez-Ruiz JM, Muñoz V (2015) Modern analysis of protein folding by differential scanning calorimetry. Methods Enzymol 567:211–233. https://doi.org/10.1016/bs.mie.2015.08.027 Min C, Geng J, Liu C, Wang Y, Huang J, Xiong YL (2024) Structural changes of flaxseed protein modified by fermentation and the gel properties and swallowing characteristics of its composite system with mung bean starch. LWT 199:116098. https://doi.org/10.1016/j.lwt.2024.116098 Karam SAE, Duconseille A, Vénien A, Ravel C, Chauvet H, Jamme F, Réfrégiers M, Astruc T (2025) Change in muscle fibre protein structure following salting process assessed by synchrotron deep UV fluorescence microspectroscopy. Food Chem 471:142801. https://doi.org/10.1016/j.foodchem.2025.142801 Karam SAE, Duconseille A, Vénien A, Ravel C, Chauvet H, Jamme F, Réfrégiers M, Astruc T (2025) Change in muscle fibre protein structure following salting process assessed by synchrotron deep UV fluorescence microspectroscopy. Food Chem 471:142801. https://doi.org/10.1016/j.foodchem.2025.142801 Jiang J, Xiong YL, Chen J (2010) pH shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions. J Agric Food Chem 58(13):8035–8042. https://doi.org/10.1021/jf1000178 Guan L, Xu H, Huang D (2011) The investigation on states of water in different hydrophilic polymers by DSC and FTIR. J Polym Res 18(4):681–689. https://doi.org/10.1007/s10965-010-9465-6 Jacobs MR, Grace M, Blumlein A, Mcmanus JJ (2019) Differential scanning calorimetry to quantify heat-induced aggregation in concentrated protein solutions. Methods in Molecular Biology 2039: 115–130. https://doi.org/10.1007/978-1-4939-9678-0_9 Bates A, Williams KM, Hagerman AE (2025) Protein thermal stability in the undergraduate biochemistry laboratory: Exploring protein thermal stability with yeast alcohol dehydrogenase. Biochem Mol Biol Educ 53(2):226–234. https://doi.org/10.1002/bmb.21880 Durowoju IB, Bhandal KS, Hu J, Carpick B, Kirkitadze M (2017) Differential scanning calorimetry — A method for assessing the thermal stability and conformation of protein antigen. J Visualized Experiments 121:55262. https://doi.org/10.3791/55262 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8463814","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":573081072,"identity":"4969d241-b6a0-41ba-8ed5-ba4037dd4137","order_by":0,"name":"Cong Min","email":"","orcid":"","institution":"Shaanxi University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Min","suffix":""},{"id":573081073,"identity":"b9ee67e5-e181-4e47-abe7-2217c66689ee","order_by":1,"name":"Yukun Wang","email":"","orcid":"","institution":"Shaanxi University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yukun","middleName":"","lastName":"Wang","suffix":""},{"id":573081074,"identity":"ef859651-7208-481f-89f4-b1b6f8f68bbf","order_by":2,"name":"Wenqian Gong","email":"","orcid":"","institution":"Shaanxi University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenqian","middleName":"","lastName":"Gong","suffix":""},{"id":573081075,"identity":"ad278ec8-13cf-40c3-bfcd-836d40a54232","order_by":3,"name":"Xiangming Ye","email":"","orcid":"","institution":"Shaanxi University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiangming","middleName":"","lastName":"Ye","suffix":""},{"id":573081077,"identity":"14fdbfa0-73bf-478e-982e-adca4efb3dfd","order_by":4,"name":"Wen Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3RsQrCMBCA4YNIXIJdI4q+QkCoCoKvklDopOAkHQQriB3UXUHwFZzEMSFQl7g7Kq4Oujmqs9LWzSHfnB9ydwCW9YdwdS/l7dFC7fVFnXgwSE8KlAi1nPl5Br7HTiZOTyoUappg7TDouMXzGGX4WCnkmhC/VAfjBiLE4ERTnpyUlVSrRqvWHE36R7ErAzWHTXICnMsr8T3QaHsUBgOj3dSEydcswzAGtycmKENCOVOvBDGTcyFbQgx/LxkVF9ij3MQkdZZqFOnb+5QORer+CAYVJ5onJx/Ib88ty7Ksr56Zp009AcVJNgAAAABJRU5ErkJggg==","orcid":"","institution":"Shaanxi University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Wen","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2025-12-28 04:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8463814/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8463814/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100369432,"identity":"caa5fd1b-c469-45e7-b26a-b80996995550","added_by":"auto","created_at":"2026-01-16 07:59:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1147085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/e2dd791a0a964154bc2e84a0.docx"},{"id":100369414,"identity":"47526421-b58d-4f41-8218-d08eec4d7e23","added_by":"auto","created_at":"2026-01-16 07:59:02","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7018,"visible":true,"origin":"","legend":"","description":"","filename":"bd91199a017940b0b76dbbc70f9b4464.json","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/ad70eafea25364a0eee59481.json"},{"id":100172491,"identity":"a0e2ba3f-603a-4002-af66-363977baedfb","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125373,"visible":true,"origin":"","legend":"","description":"","filename":"bd91199a017940b0b76dbbc70f9b44641enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/1354f77e9ca0e69f5efde2da.xml"},{"id":100172485,"identity":"f3cbd7ed-4c79-4015-aef8-061e1b573994","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":430513,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/9f0ed70080425d4e0c537c0a.png"},{"id":100172488,"identity":"0d13c645-7468-4663-a285-3d5c00c7465d","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":308474,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/75cb2892fe7b39e34d2f1304.jpeg"},{"id":100368258,"identity":"1bc971c0-003a-40a7-88f5-1e6eaff6e596","added_by":"auto","created_at":"2026-01-16 07:57:47","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85099,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/f4ad59a5c12594c553c98920.jpeg"},{"id":100172484,"identity":"15a14d66-17ec-4bd3-a719-1f6fc22e431d","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44663,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/376168900ec493bcd17d9c87.jpeg"},{"id":100172493,"identity":"ffc035a5-4f14-463c-8106-086d9dc3ceb1","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":262434,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/6088ce1808c9f9e7a128b76d.png"},{"id":100172494,"identity":"98552e6d-49b6-45b2-bcbf-8c766543d5d4","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113098,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/9f73635f112ab34aa07f5624.png"},{"id":100369159,"identity":"7ae79ca2-7ab0-4e32-8848-535fefb82f74","added_by":"auto","created_at":"2026-01-16 07:58:44","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61910,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/80b1c5ab6388490252e1c58a.png"},{"id":100368324,"identity":"ae463910-03eb-4d9b-839c-5796b1b966c3","added_by":"auto","created_at":"2026-01-16 07:57:51","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46152,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/5a2fed14feb37602f2b12843.png"},{"id":100368861,"identity":"68a37125-b686-4643-94a1-4645c8a1dd93","added_by":"auto","created_at":"2026-01-16 07:58:27","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22845,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/a57368c631bd5fe25c202a50.png"},{"id":100172495,"identity":"7f3d06c3-95a4-4e23-b8a0-c9a2d1f8086f","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63001,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/55c4350b022ad9bc0fd76e80.png"},{"id":100172497,"identity":"118adb64-c6c7-4085-b13c-1482a54b8da3","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124128,"visible":true,"origin":"","legend":"","description":"","filename":"bd91199a017940b0b76dbbc70f9b44641structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/23f194e5f213947afd753798.xml"},{"id":100368770,"identity":"6126fb88-4529-4a8a-82b2-36bbfe274633","added_by":"auto","created_at":"2026-01-16 07:58:20","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135545,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/2338f8785b7434794c4305d9.html"},{"id":100172480,"identity":"7b43787a-ff72-4719-a1e5-1c434c0856ec","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111476,"visible":true,"origin":"","legend":"\u003cp\u003eImages and microstructure of FFP after different fermentation stages. 0 h-10 h: A1-E1: 2000×\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/1ad8a125ef61e7ce4392433a.jpg"},{"id":100172481,"identity":"1b0c21af-5574-467d-ab41-ab31400717ad","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61910,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE results of the FFP following different fermentation\u003cdel\u003e \u003c/del\u003eperiods.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/049ce891b0799e39bb37d130.png"},{"id":100369103,"identity":"e5745b46-6671-4f78-82b6-2e0e44e6dc1d","added_by":"auto","created_at":"2026-01-16 07:58:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77855,"visible":true,"origin":"","legend":"\u003cp\u003eUV absorption spectrogram of the FFP after different fermentation durations.(A) The original UV absorption spectrogram; (B) The deconvoluted UV absorption spectrogram.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/8f2ed2d38142012d0e057b3e.png"},{"id":100172482,"identity":"71c22d90-f210-40e4-b7e4-21cd8b1a5d16","added_by":"auto","created_at":"2026-01-13 17:04:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22845,"visible":true,"origin":"","legend":"\u003cp\u003eDSC curve of the FFP following various fermentation periods.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/efe6af845a7c4344653e213a.png"},{"id":100369183,"identity":"180dc5f2-85e7-4aed-af2a-d9a9f68a96a4","added_by":"auto","created_at":"2026-01-16 07:58:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63001,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the structure modified by fermentation of flaxseed protein.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/079834039e628e8fd4047758.png"},{"id":104403735,"identity":"ee4ffb71-540b-47f4-b80e-b7cd4096495b","added_by":"auto","created_at":"2026-03-11 12:18:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1170411,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8463814/v1/f6271cf0-43f5-452d-8a19-d42f1fc3bbd8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural and functional evolution of flaxseed protein during probiotic fermentation: Insights into proteolytic degradation, conformational rearrangement, and thermal property","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFlaxseed protein (FP), recognized as a high-quality plant-based protein, has garnered significant attention in the realm of health foods due to its substantial nutritional value and functional properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is abundant in amino acids, encompassing 8 essential amino acids that constitute over 30% of its composition, and shares a comparable amino acid profile and nutritional attributes with soy protein. FP is particularly rich in aspartic acid, glutamic acid, and arginine; moreover, the content of seven essential amino acids, including phenylalanine, isoleucine, lysine, leucine, threonine, valine, and tryptophan, exceeds the adult recommended intake by the World Health Organization [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Compared to soy protein, FP boasts a higher arginine content and a lower lysine/arginine ratio, which positively influences muscle and immune function as well as cardiovascular health in the elderly, particularly those with swallowing difficulties. Beyond its role as a nutritional supplement, FP also holds the potential to enhance the functional properties of food [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It can serve as a texture regulator to enhance the hardness, elasticity, color, appearance, and flavor of baked goods or pastries such as bread, pizza, and biscuits. Additionally, FP can improve the viscoelasticity and water retention of snacks or cereal products, impart a unique hardness and viscosity to noodle products like pasta, and decrease cooking losses in meat products such as sausages [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nevertheless, natural FP faces limitations such as poor solubility, low thermal stability, and limited functionality, which severely impede its application in high-value products [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, microbial fermentation modification has emerged as a strategy for enhancing plant protein quality, owing to its environmentally friendly and efficient nature [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous research has demonstrated that microbial fermentation methods, including those using Lactobacillus, Lactobacillus rhamnosus, and Lactobacillus plantarum, can effectively eliminate toxic components such as cyanogenic glycosides from flaxseed. These methods also influence the formation of biogenic amines and volatile compounds in flaxseed [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For functional proteins, employing microbial strains like Lactobacillus plantarum, Bacillus subtilis, and Aspergillus oryzae for fermentation can break down protein molecules, resulting in a large number of small molecule peptides. Many of these peptides possess various physiological activities, such as inhibiting angiotensin-converting enzyme, antioxidant properties, antibacterial effects, and anti-diabetic potential, indicating their potential as bioactive peptides [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, the digestibility and thermal stability of FP fermented with probiotics can be regulated [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Consequently, microbial fermentation holds significant promise for improving the functional properties of FP and equivalent plant proteins, as well as their application in the functional foods domain. However, research on its specific mechanisms of action and processes of change remains limited.\u003c/p\u003e \u003cp\u003eIn this study, we innovatively used probiotic complexes (Lactobacillus bulgaricus: Bifidobacterium lactis\u0026thinsp;=\u0026thinsp;1:2) for fermentation modification of FP. Based on preliminary experiments, it was found that the two bacteria had a synergistic effect under this ratio, which could more efficiently hydrolyze FP macromolecular proteins, and the degree of thermal stability reduction was significantly better than that of single strain fermentation. The structural evolution of fermented flaxseed protein (FFP) during the first 10 h of fermentation was systematically investigated using laser particle size analysis, scanning electron microscopy (SEM), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet spectroscopy, and differential scanning calorimetry (DSC). This research provides a scientific foundation for the precise regulation of fermentation processes and promotes the efficient utilization of plant protein resources by establishing a multidimensional correlation map of structure, thermal performance, and function.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and chemical reagent\u003c/h2\u003e \u003cp\u003eFP (\u0026ge;\u0026thinsp;81% protein, 3.62% moisture, 5.24% lipids) was obtained from Shaanxi Kangduopu Biotechnology Co., Ltd. (Xi'an, Shaanxi, China). \u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e and \u003cem\u003eBifidobacterium lactis\u003c/em\u003e (both with a viable count of 210\u0026sup1;⁰ cfu) were sourced from Shaanxi Benhe Bioengineering Co., Ltd. (Xi'an, Shaanxi, China). Water is deionized. All other reagents were analytical grade reagents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Fermentation of flaxseed protein\u003c/h2\u003e \u003cp\u003eWeigh 30.0 g of FP powder add deionized water to prepare a suspension. Stir the suspension magnetically at room temperature for 10 minutes, then dilute it to 100 mL to achieve a final concentration of 30% FP suspension. The FP suspension was sterilized in a 95 ℃ water bath for 10 minutes and subsequently cooled rapidly to 37 ℃. Following the method of Xu et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], probiotic fermentation agents (1% \u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e and 2% \u003cem\u003eBifidobacterium lactis\u003c/em\u003e, both with a viable cell count of 2\u0026times;10\u0026sup1;⁰ cfu) were added to the FP suspension and fermented at 37\u0026deg;C for 2, 4, 6, 8, and 10 h for degradation. The unfermented natural FP (0 h) served as the control group. Fermentation - degradation FP samples with different fermentation times were frozen overnight at -80 ℃, followed by freeze - drying at -80 ℃ for 48 h. The dried samples were then ground into powder for subsequent use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Particle size determination\u003c/h2\u003e \u003cp\u003eFreeze-dried FFP powder samples with varying fermentation times were added to deionized water to prepare a solution with a concentration of 4 mg/mL. Magnetic stirring was performed for 2 h to ensure complete dissolution of the protein and uniform dispersion of the particles. The size of the protein particles was measured using a laser nanoparticle size analyzer. The parameters were as follows: the dispersant was a 10 mmol/L phosphate buffer solution (pH 7.0), and the dispersant refractive index was 1.3318. The measurement was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Scanning electron microscopy\u003c/h2\u003e \u003cp\u003eThe method described by Min et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was followed with minor modifications. fine adjustments were made by fixing freeze-dried FFP powder samples with varying fermentation times on a sample stage and performing a gold spraying treatment on the samples. Subsequently, observation and photography were conducted using a scanning electron microscope at a voltage of 20 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Protein electrophoresis detection\u003c/h2\u003e \u003cp\u003eDissolve freeze-dried powder samples of FFP with varying fermentation times in deionized water, stir magnetically at room temperature for 10 minutes, filter through a 0.45 \u0026micro;m filter, and prepare an FFP solution with a concentration of 1 mg/mL. Add an appropriate amount of SDS-PAGE loading buffer, mix well, boil at 95 ℃ for 5 minutes, and immediately place on ice; centrifuge at 12,000 rpm at 4 ℃ for 10 minutes. Then, take 20 \u0026micro;L of each supernatant for electrophoresis separation (with a concentrated gel concentration of 4% and a separated gel concentration of 15%), applying a constant voltage of 80 V in the concentrated gel area and 120 V in the separated gel area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. UV full wavelength scanning detection\u003c/h2\u003e \u003cp\u003ePrepare a solution with a mass concentration of 2.0 mg/mL using FFP freeze-dried powder for testing. Transfer approximately 3 mL to a quartz colorimetric dish and use a UV-visible spectrophotometer to perform a full wavelength UV scan. Set the scanning wavelength range from 200 to 600 nm, with a slit width of 1.0 nm and a sampling interval of 0.5 nm, and conduct the scan at a slow speed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Thermal characteristic analysis\u003c/h2\u003e \u003cp\u003eTake 3.0 mg samples of freeze-dried FFP powder with varying fermentation times, seal them in an aluminum crucible, and analyze using a differential scanning calorimeter. Set the instrument parameters as follows: a heating rate of 10.0 ℃/min, within a temperature range of 80 ℃ to 180 ℃. Record the detection values: starting temperature (To), peak temperature (Tp), final temperature (Tc), and enthalpy value (ΔH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Data analysis\u003c/h2\u003e \u003cp\u003eAll tests were conducted in triplicate, and the data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A one-way analysis of variance (ANOVA) and Duncan's test were used to perform the statistical analysis with SPSS 22 software (IBM, NY, USA). A \u003cem\u003eP-value of\u003c/em\u003e less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Particle size analysis of fermented flaxseed protein\u003c/h2\u003e \u003cp\u003eThe laser particle size analysis results indicated that the particle size distribution of FFP during the fermentation process exhibited significant time-dependent evolution characteristics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At the unfermented stage (0 h), the sample particle size distribution was dominated by large particles (\u0026ge;\u0026thinsp;100 \u0026micro;m, accounting for 25.71%) and medium particles (10\u0026ndash;100 \u0026micro;m, accounting for 44.02%). As the fermentation process progressed (2\u0026ndash;10 h), the proportion of particles with a diameter greater than 100 \u0026micro;m sharply decreased to 3.20% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the proportion of particles with a diameter between 10\u0026ndash;100 \u0026micro;m significantly increased to 64.1%. It is noteworthy that the proportion of \u0026lt;\u0026thinsp;0.1 \u0026micro;m ultrafine particles decreased from 5.96% to 2.38%, suggesting that degradation products may migrate to larger particle size ranges through dissolution or recombination mechanisms [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The dynamic evolution of particle size homogenization distribution during fermentation reveals a two-stage mechanism of action [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. On one hand, the protease-dominated primary degradation stage (0\u0026ndash;4 h) rapidly reduces large particles (100 \u0026micro;m). This process originates from the specific hydrolysis of peptide bonds by endonucleases secreted by probiotic fermentation agents (Lactobacillus bulgaricus and Bifidobacterium lactis) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. On the other hand, there is a secondary aggregation stage of degradation products (6\u0026ndash;10 h), where the proportion of medium particles (10\u0026ndash;100 \u0026micro;m) continues to increase. The possible pathway for its formation is the formation of hydrophobic core aggregates and charged groups through electrostatic interactions after the exposure of hydrophobic peptide segments to construct a stable network structure [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticle size of the FFP following various fermentations. Means with different letters (a-f) differ significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) among the data in the same column\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.1 \u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1-1 \u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;10 \u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026ndash;100 \u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100 \u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Microstructure of fermented flaxseed protein\u003c/h2\u003e \u003cp\u003eDuring the fermentation process, the appearance of FFP powder exhibits regular changes over time (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the early stage of fermentation (0 h), the powder has a lighter color, uneven texture, and a tight structure with obvious clumping. As the fermentation time is extended to 2\u0026ndash;4 h, the color of the powder gradually deepens, the texture begins to become loose, and the phenomenon of clumping gradually decreases. In the middle stage of fermentation (6\u0026ndash;8 h), the color further turns brown due to Maillard reaction or accumulation of fermentation products, appearing light brown, with a significantly loose texture, and the particle size decreases and gradually becomes uniform. In the late stage of fermentation (10 h), the powder color is the darkest, close to burnt yellow or brown, and the particle size gradually becomes uniform, consistent with the data of particle size. The overall trend indicates that the longer the fermentation time, the darker the powder color, the looser the texture, and the gradually improved structural uniformity.\u003c/p\u003e \u003cp\u003eUpon SEM observation, it was revealed that the microstructure of FFP undergoes significant stage changes throughout the fermentation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Prior to fermentation (at 0 h), FFP displays a dense, block-like or layered structure with a smooth surface, suggesting that its native conformation remains intact and intermolecular forces, (such as hydrogen bonding and hydrophobic interactions, are stable. In the initial stage of fermentation (2\u0026ndash;4 h), the sample's particle size ranges from 50\u0026ndash;100 \u0026micro;m, indicating that the probiotic fermentation agents (Lactobacillus bulgaricus and Bifidobacterium lactis) have begun to act on peptide bonds, resulting in localized structural depolymerization. During the intermediate stage of fermentation (6\u0026ndash;8 h), FFP becomes further loosened, potentially due to intermolecular cross-linking caused by the exposure of hydrophobic groups, (uch as phenylalanine and leucine residues. In the late stage of fermentation (10 h), the microstructure is completely fragmented, presenting dispersed particles with a size of less than 50 \u0026micro;m [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The findings indicate that fermentation transforms FFP from a dense block structure into nano-sized dispersed particles, a process dominated by the degradation of probiotic fermentation agents and achieving function-oriented recombination through hydrophobic electrostatic equilibrium. As fermentation time progresses, the particle size of FFP powder becomes increasingly uniformly distributed [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This process is closely linked to the degradation of proteins, hydrophobic interactions, and structural stability changes of probiotic fermentation agents, offering a structural basis for their functional modification (such as active peptide release and processing performance optimization).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. The effect of fermentation degradation on the relative molecular weight of flaxseed protein\u003c/h2\u003e \u003cp\u003eThe molecular weight distribution of peptide segments in fermentation products is another important indicator reflecting the extent of protein fermentation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This article analyzed the molecular weight distribution of peptide segments in FFP after degradation at different times using the SDS-PAGE method, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. During fermentation, it was revealed that unfermented FFP (0 h) exhibited significant bands in the molecular weight\u0026thinsp;\u0026gt;\u0026thinsp;100 kDa region, indicating its natural state as high molecular weight aggregates or complexes. As the fermentation time extends (2\u0026ndash;10 h), the intensity of the bands greater than 100 kDa diminishes progressively, while there is a corresponding rise in the intensity of bands within the 15\u0026ndash;35 kDa low molecular weight range. The degradation process of FFP exhibits marked time-dependent characteristics [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the early stage of fermentation (0\u0026ndash;4 h), rapid degradation occurs, and the content of \u0026gt;\u0026thinsp;100 kDa protein decreases within 4 h, indicating that the probiotic fermentation agent is rapidly activated in the early stage of fermentation, preferentially attacking the loose structural domains of FFP (such as the α-helix region). There is a dynamic equilibrium during the mid-fermentation stage (4\u0026ndash;8 h), during which the degradation rate slows down, possibly due to the remaining substrate being a resistant structure (β-folding cluster or disulfide bond stable region), with hydrophobic core exposure driving aggregation and forming stable secondary structures [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the final stage of fermentation, most of the proteins are depolymerized into small peptides, maximizing their functional activity. Unfermented FP exists in the form of oligomers (180 kDa), while smaller fragments of 10\u0026ndash;15 kDa gradually increase with fermentation time after fermentation, indicating that the dissociation of non-covalent bonds (hydrophobic interactions, etc.) dominates subunit separation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The SDS-PAGE experimental results further confirmed the observation results of particle size analysis and scanning electron microscopy, indicating that with the prolongation of fermentation time, the structure of FFP undergoes further dissociation, gradually forming more uniform and smaller particles. This study provides a theoretical basis for the high-value utilization of FP and lays a technical foundation for precise regulation of fermentation processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 UV absorption spectrum of fermented flaxseed protein\u003c/h2\u003e \u003cp\u003eThe FFP sample was scanned across the full wavelength range from 200 nm to 600 nm using a UV spectrophotometer, and its UV absorption spectrum is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In the UV absorption spectrum, the absorption peak near 280 nm is primarily attributed to aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). The absorbance at this wavelength in the spectrum markedly decreases with fermentation time (0 h \u0026minus;\u0026thinsp;10 h), suggesting that protein degradation may be occurring, resulting in the release of aromatic amino acids or structural damage. The overall shape of the absorbance curve, including peak width and peak position, shifts at various fermentation time points, which may reflect changes in the conformation of protein secondary structures, (such as alpha helices and beta sheets [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. During the late stage of fermentation (6\u0026ndash;10 h), the absorbance stabilized compared to the control group (0 h), suggesting that the protein structure might have reached a dynamic equilibrium or that microbial metabolic activity had slowed.\u003c/p\u003e \u003cp\u003eUV-visible spectroscopy analysis reveals that the structural characteristics of FFP undergo significant dynamic evolution throughout the fermentation process. The absorbance at 280 nm decreases as fermentation time (extends from 0 to 10 h, indicating the release or structural damage of aromatic amino acids (tryptophan, tyrosine) due to protein degradation. This is consistent with the gradual disappearance of high molecular weight bands in SDS-PAGE. Additionally, the spectral peak position in the 250\u0026ndash;300 nm range shifts from 275 nm to 282 nm, suggesting a transition from an ordered to a disordered protein secondary structure, potentially driven by molecular rearrangement induced by the exposure of hydrophobic groups. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The UV absorption spectroscopy results indicate that the fermentation modification of FFP involves protease-mediated degradation and conformational relaxation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Thermal characteristics of FFP fermented at different times\u003c/h2\u003e \u003cp\u003e​The thermal stability of proteins is an important factor affecting the processing quality of gel food produced through heat treatment [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To comprehensively evaluate the influence and function of probiotic fermentation on the thermal characteristics of proteins, this study conducted DSC analysis on FFP at different fermentation stages. The DSC thermograms revealed significant changes in the thermal characteristics of FFP throughout the fermentation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The fermentation of probiotics markedly diminishes the thermal stability of FFP, evidenced by a reduction in the denaturation temperature and an increase in structural heterogeneity. This phenomenon is closely associated with protein degradation, the destruction of the secondary structure, and the exposure of hydrophobic groups [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The unfermented sample (0 h) displayed a single endothermic peak at 149.27\u0026thinsp;\u0026plusmn;\u0026thinsp;8.22\u0026deg;C, indicating that natural FP has high thermal stability and an ordered secondary structure. Its tightly folded secondary structures, comprising alpha helices and beta folds, require high energy for melting. As fermentation progresses (2\u0026ndash;10 h), the primary endothermic peak gradually shifts to lower temperatures (10 h: 101.77\u0026thinsp;\u0026plusmn;\u0026thinsp;8.69\u0026deg;C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a decrease in denaturation temperature suggests the disruption of stable interactions, (such as hydrogen bonding and hydrophobic stacking. During the fermentation process, which lasts 2 to 10 h, the peak position shifts towards lower temperatures. This shift indicates a loosening of the protein structure, potentially leading to a weakening of intramolecular forces, such as hydrogen bonding and hydrophobic interactions (Dickson et al., 2025). According to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, as fermentation progresses, the starting temperature (To) of FFP decreases from 132.75\u0026deg;C (at 0 h) to 101.77\u0026deg;C (at 10 h), a decrease of 23.3%, indicating a reduction in the energy required for fermentation to disrupt the protein crystal structure. The peak temperature (Tp) decreases from 149.27\u0026deg;C (at 0 h) to 115.43\u0026deg;C (at 10 h), a decrease of 22.7%, reflecting a reduction in the orderliness and thermal stability of FFP molecules. The termination temperature (Tc) decreases from 170.51\u0026deg;C (at 0 h) to 129.99\u0026deg;C (at 10 h), a decrease of 23.8%, indicating that the gelatinization process is accelerated and the efficiency of molecular chain disentanglement is improved. The enthalpy change (ΔH) continues to increase, rising from 16.77 J/g at 0 h to 22.80 J/g at 10 h\u0026mdash;an increase of 36%. This suggests that the gelatinization process requires more heat absorption. It indicates that the crystal area is damaged or the disordered structure has increased, which in turn suggests that its water absorption is enhanced. The results of DSC and UV detection of peak displacement and structural disorder confirmed each other, indicating that fermentation modified FFP is more suitable for gel food.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOnset temperature (To), peak temperature (Tp), concluding temperature (Tc), and enthalpy (ΔH) of the FFP after various fermentation. Means with different letters (a-e) differ significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) among the data in the same column.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003eO\u003c/sub\u003e (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003csub\u003eP\u003c/sub\u003e (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003csub\u003eC\u003c/sub\u003e (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔH (J/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e149.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e170.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e121.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e139.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e163.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e111.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e129.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e107.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e141.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e104.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e132.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e129.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Schematic mechanism\u003c/h2\u003e \u003cp\u003eUnder the influence of probiotic fermentation, where L. bulgaricus and B. lactis maintain a symbiotic relationship at a ratio of 1:2, the structure and molecular forces of FP undergo significant changes, particularly during the critical transition period of 8\u0026ndash;10 h. Based on the molecular weight and thermal characteristics of flaxseed protein (FP) fermented over various periods, this study proposes a mechanism logic model of \"microbial protease hydrolysis, structure recombination, and performance alteration\" (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study achieved a probiotic-driven structural reconstruction of FP through the regulation of fermentation using Lactobacillus bulgaricus and Bifidobacterium lactis. Time characterization revealed a three-stage transformation mechanism. Within 0\u0026ndash;4 h, microbial proteases preferentially hydrolyze hydrophobic peptide bonds, thereby reducing large molecular aggregates (\u0026gt;\u0026thinsp;100 \u0026micro;m; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and cleaving high molecular weight proteins (\u0026gt;\u0026thinsp;100 kDa), as indicated by particle size distribution and SDS-PAGE analysis. Subsequently, hydrophobic electrostatic recombination (4\u0026ndash;8 h) formed a metastable porous network. The red shift of the UV absorption peak at A280 indicates that the secondary structure is disordered. The final fermentation stage, which lasted 8\u0026ndash;10 h, resulted in uniform sub-50 \u0026micro;m particles. Additionally, the DSC results indicated that the gel transition temperature decreased by 23.3\u0026ndash;23.8%, and the ΔH increased by 36%, signifying a significant alteration in its thermal behavior. This protease-mediated cascade reaction, which converts ordered aggregates into disordered particle systems, provides a feasible strategy to reduce the restrictions of FP in the food preparation process due to high deformation temperatures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study examined the dynamic regulatory mechanisms of probiotic fermentation (Lactobacillus bulgaricus and Bifidobacterium lactis) on the structure and thermal properties of FP using a multi-scale characterization system, and established a multidimensional evaluation system covering particle size, molecular weight, thermal characteristics, and conformation. The results showed that the particle size of FP evolved in a time-dependent manner: within 0-4 h of fermentation, protease specific hydrolysis of peptide bonds led to a sharp decrease in the proportion of particles larger than 100 μ m, while the proportion of particles larger than 10-100 μ m increased. SDS-PAGE results showed the degradation of polymer aggregates (\u0026gt;100 kDa) accompanied by the release of aromatic amino acids; Fermentation for 4-8 h enters the hydrophobic driven recombination stage, forming a stable network through hydrophobic electrostatic equilibrium; At 10 h, FP completely dissociated into dispersed particles\u0026lt;50 μ m (SEM verification). In terms of structure, the UV spectrum shows a decrease in absorbance and a red shift at 280 nm, indicating a decrease in the orderliness of the secondary structure; The DSC results further indicate that fermentation reduces the thermal denaturation temperature (To, Tp, Tc) and increases ΔH, indicating that hydrogen bonding and hydrophobic interactions are disrupted, leading to crystal structure collapse and increased disorder, requiring gelatinization to absorb more heat.\u003c/p\u003e\n\u003cp\u003eThis study clarifies that probiotic fermentation destroys the ordered structure of FP through enzymolysis, reduces its thermal stability and improves its heat absorption capacity. This feature is highly consistent with the process requirements of plant based meat products for low heat treatment and high gelatinization, which is expected to improve the caking and texture problems of traditional FP in thermal processing. In the future, we can focus on optimizing the application ratio of fermented FP in plant-based meat, and combine molecular simulation to deeply analyze the structure-activity relationship of key peptide segments, in order to achieve precise regulation and industrial application of fermentation technology.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest regarding the publication of this paper, and the manuscript is approved by all authors for publication.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe financial support from the Scientific Research Program Funded by Education Department of Shaanxi Provincial Government (Program No. 24JK0371), the Scientific Research Project for Talented Scholars of Shaanxi University of Technology (SLGRC202407, SLGRCQD2203), National Natural Science Foundation of China Cultivation Project (SLGGZRPY14).\u003c/p\u003e\u003ch2\u003eAuthor Contribaution\u003c/h2\u003e\u003cp\u003eCong Min : Conceptualization, Methodology, Formal analysis, Funding acquisition, Resources, Supervision, Writing \u0026ndash; original draft. Yukun Wang : Methodology, Investigation, Software, Formal analysis, Data curation. Wenqian Gong: Methodology, Resources. Xiangming Ye : Formal analysis, Methodology. Wen Su : Conceptualization, Resources, Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData availability： Data will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang B, Han J, Liu C, Zhang J, Qi Y (2025) Flaxseed protein content prediction based on hyperspectral wavelength selection with fractional order ant colony optimization. Front Nutr 12:1551029. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnut.2025.1551029\u003c/span\u003e\u003cspan address=\"10.3389/fnut.2025.1551029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen J, Fu J, Zhao X (2024) Structural characterization and performance analysis of flaxseed isolate protein. Trans Chin Soc Agricultural Eng 40(5):326\u0026ndash;336. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.11975/j.issn.1002-6819.202312060\u003c/span\u003e\u003cspan address=\"10.11975/j.issn.1002-6819.202312060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaroov\u0026aacute; M, Roudnick\u0026aacute; P, B\u0026aacute;rta J, Zdr\u0026aacute;hal Z, B\u0026aacute;rtov\u0026aacute; V, Stupkov\u0026aacute; A, Lorenc F, Bjelkov\u0026aacute; M, Kyselka J, Jaroov\u0026aacute; E (2024) Proteomic profile of flaxseed (Linum usitatissimum L.) products as influenced by protein concentration method and cultivar. Foods 13(9):1288. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods13091288\u003c/span\u003e\u003cspan address=\"10.3390/foods13091288\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKajla P, Sharma A, Sood DR (2015) Flaxseed\u0026mdash;a potential functional food source. J Food Sci Technol 52(4):1857\u0026ndash;1871. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13197-014-1293-y\u003c/span\u003e\u003cspan address=\"10.1007/s13197-014-1293-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma M, Saini CS (2022) Amino acid composition, nutritional profiling, mineral content and physicochemical properties of protein isolate from flaxseeds (Linum usitatissimum). J Food Meas Charact 16(1):38\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11694-021-01221-0\u003c/span\u003e\u003cspan address=\"10.1007/s11694-021-01221-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabetafika HN, Van Remoortel V, Danthine S, Paquot M, Blecker C (2011) Flaxseed proteins: Food uses and health benefits. Int J Food Sci Technol 46(2):221\u0026ndash;228. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2621.2010.02477.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2621.2010.02477.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImran S, Munir S, Altemimi AB, Fatima I, Rabail R, Batool I (2024) Therapeutic implications of flaxseed peptides and bioactive components against various diseases. J Funct Foods 119:106324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2024.106324\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2024.106324\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaushik P, Dowling K, McKnight S, Barrow CJ, Wang B, Adhikari B (2016) Preparation, characterization and functional properties of flax seed protein isolate. Food Chem 197:212\u0026ndash;220. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2015.09.106\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2015.09.106\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD'Almeida AP, Neta AAI, de Andrade Lima MATL (2024) Plant-based probiotic foods: Current state and future trends. Food Sci Biotechnol 33(15):3401\u0026ndash;3422. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10068-024-01533-z\u003c/span\u003e\u003cspan address=\"10.1007/s10068-024-01533-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Xu Y, Song S, Zhan S, Li X, Wang H, Zhu Z, Yan L, Peng Y, Xie C (2024) Effect of different probiotic fermentations on the quality of plant-based hempseed fermented milk. Foods 13(24):4076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods13244076\u003c/span\u003e\u003cspan address=\"10.3390/foods13244076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartkiene E, Schleining G, Juodeikiene G, Vidmantiene D, Krungleviciute V (2014) The influence of lactic acid fermentation on biogenic amines and volatile compounds formation in flaxseed and the effect of flaxseed sourdough on the quality of wheat bread. LWT-Food Sci Technol 56(2):445\u0026ndash;450. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2013.12.006\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2013.12.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, Wang L, Li D, Wang Y, Mao ZH, Chen XD (2012) The digestibility and thermal properties of fermented flaxseed protein. International Journal of Food Engineering 8(4): Article 23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/1556-3758.2178\u003c/span\u003e\u003cspan address=\"10.1515/1556-3758.2178\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Liu X, Li S, Fang Y, Cao Y (2025) Formation and morphology of flaxseed protein isolate amyloid fibrils as governed by NaCl concentration. Food Hydrocolloids 166:111300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodhyd.2025.111300\u003c/span\u003e\u003cspan address=\"10.1016/j.foodhyd.2025.111300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbe-Slabi S, Mesieres O, Beaubier S, Beau L, Aymes A, Roques-Carmes T, Sadtler V, Kapel R (2025) The role of extraction and purification conditions in flaxseed protein isolate production and its emulsifying properties. LWT-Food Sci Technol 215:117170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2024.117170\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2024.117170\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePihlanto A, Johansson T, M\u0026auml;kinen S (2012) Inhibition of angiotensin I-converting enzyme and lipid peroxidation by fermented rapeseed and flaxseed meal. Eng Life Sci 12(4):450\u0026ndash;456. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/elsc.201100137\u003c/span\u003e\u003cspan address=\"10.1002/elsc.201100137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, Wang L, Li D, Wang Y, Mao ZH, Chen XD (2012) The digestibility and thermal properties of fermented flaxseed protein. International Journal of Food Engineering 8(4): Article 23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/1556-3758.2178\u003c/span\u003e\u003cspan address=\"10.1515/1556-3758.2178\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Cui H, Xu J, Yuan Z, Liu X, Fan X, Li J, Zhu D, Liu H (2022) Effects of different probiotic fermentations on the quality, soy isoflavone and equol content of soy protein yogurt made from soy whey and soy embryo powder. LWT-Food Sci Technol 157:113096. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2022.113096\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2022.113096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin C, Wang Y, Li Y, Zhu Z, Li M, Chen W, Yi J, Liu M, Feng L, Cao Y (2025) Effects of transglutaminase on the gelation properties and digestibility of pea protein isolate with resonance acoustic mixing pretreatment. Food Chem 469:142534. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2024.142534\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2024.142534\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Peng C, Chen X, Imran M, Zhang H, Sakandar HA (2023) Impact of fermentation on antinutritional factors and protein degradation of legume seeds: A review. Food Reviews Int 39(3):1227\u0026ndash;1249. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/87559129.2021.1931300\u003c/span\u003e\u003cspan address=\"10.1080/87559129.2021.1931300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToure M, Crews CM (2016) Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew Chem Int Ed 55(6):1966\u0026ndash;1973. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.201507978\u003c/span\u003e\u003cspan address=\"10.1002/anie.201507978\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei Y, Hinchliffe BA, Minelli C (2021) Measurement of the size distribution of multimodal colloidal systems by laser diffraction. ACS Omega 6(22):14049\u0026ndash;14058. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsomega.1c00411\u003c/span\u003e\u003cspan address=\"10.1021/acsomega.1c00411\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi P, Xie C, Zeng Q, Yuan Y (2023) Effect of different hydrophobic soybean isolated peptides and their zinc complexes on the growth and fermentation of Lactobacillus bulgaricus. Int J Food Sci Technol 58(12):6411\u0026ndash;6420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ijfs.16752\u003c/span\u003e\u003cspan address=\"10.1111/ijfs.16752\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Fei Y, Li C, Cheng J, Xue F (2024) Impact of probiotic fermentation on the physicochemical properties of hemp seed protein gels. Polymers 16(21):3032. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/polym16213032\u003c/span\u003e\u003cspan address=\"10.3390/polym16213032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumari K, Kashyap P, Chakrabarti P (2023) Germination and probiotic fermentation: A way to enhance nutritional and biochemical properties of cereals and millets. Food Sci Biotechnol 33(3):505\u0026ndash;518. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10068-023-01401-2\u003c/span\u003e\u003cspan address=\"10.1007/s10068-023-01401-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira RPS, Florence ACR, Perego P, Oliveira MN, Converti A (2011) Use of lactulose as prebiotic and its influence on the growth, acidification profile and viable counts of different probiotics in fermented skim milk. Int J Food Microbiol 148(2):111\u0026ndash;117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2010.11.011\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2010.11.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawson EQ, Sadler AJ, Harmatz D, Brandau DT, Micanovic R, MacElroy RD, Middaugh CR (1981) A simple experimental model for hydrophobic interactions in proteins. Biochemistry 20(14):3995\u0026ndash;4000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/npg.els.0002975\u003c/span\u003e\u003cspan address=\"10.1038/npg.els.0002975\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFragneto G, Su TJ, Lu JR, Thomas RK, Rennie AR (2000) Adsorption of proteins from aqueous solutions on hydrophobic surfaces studied by neutron reflection. Phys Chem Chem Phys 2(22):5214\u0026ndash;5221. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/B004221H\u003c/span\u003e\u003cspan address=\"10.1039/B004221H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang X, Ye H, Chen MD, Chen XD, Chen MH, Chen JY, Kong WH, Wang QQ, Zhang ZY (2024) Effects of co-fermentation of lactobacillus plantarum and inulin on beany flavor and physicochemical quality of soy yogurt in a multiple lactic acid bacteria fermentation system. Cereal Chem 101(1):248\u0026ndash;262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cche.10741\u003c/span\u003e\u003cspan address=\"10.1002/cche.10741\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTindjau R, Chua J-Y, Liu SQ (2023) Growth, substrate, and metabolite changes of probiotic Bifidobacterium animalis subsp. lactis in soy (tofu) whey. Fermentation 9(12):1024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/fermentation9121024\u003c/span\u003e\u003cspan address=\"10.3390/fermentation9121024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Q, Liu L, Wang L, Yang R, Liu X, Dong Y, Zeng X, Liu X, Du Q, Wu Z, Pan D (2025) Nanocoating of quinoa protein and hyaluronic acid enhances viability and stability of Limosilactobacillus fermentum RC4 microcapsules. Int J Biol Macromol 307(Pt 1):141863. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2025.141863\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2025.141863\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKravchenko IV, Furalyov VA, Pshennikova ES, Fedorov AN, Popov VO (2024) The effect of fermentation by lactobacilli on the functional\u0026ndash;technological properties of pea protein isolates. Appl Biochem Microbiol 60(6):1388\u0026ndash;1397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1134/S0003683824605493\u003c/span\u003e\u003cspan address=\"10.1134/S0003683824605493\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoss PD, Rekharsky MV (1996) Thermodynamics of hydrogen bond and hydrophobic interactions in cyclodextrin complexes. Biophys J 70(3):1231\u0026ndash;1239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0006-3495(96)79415-8\u003c/span\u003e\u003cspan address=\"10.1016/S0006-3495(96)79415-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArai T, Mio K, Onoda H, Chavas LMG, Umena Y, Sasaki YC (2023) The blinking of small-angle X-ray scattering reveals the degradation process of protein crystals at microsecond timescale. Int J Mol Sci 24(23):16640. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms242316640\u003c/span\u003e\u003cspan address=\"10.3390/ijms242316640\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei G, Regenstein JM, Zhou P (2021) The fermentation-time dependent proteolysis profile and peptidomic analysis of fermented soybean curd. J Food Sci 86(11):4182\u0026ndash;4192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1750-3841.15823\u003c/span\u003e\u003cspan address=\"10.1111/1750-3841.15823\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Y, Yang Q, Wang M, He W, Dai W, Tang X (2024) Flavor and nutritional characteristics of surimi product with lactiplantibacillus plantarum as a reinforcing starter culture. Food Bioscience 62:105229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fbio.2024.105229\u003c/span\u003e\u003cspan address=\"10.1016/j.fbio.2024.105229\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZotta T, Piraino P, Ricciardi AM, McSweeney PLH, Parente E (2006) Proteolysis in model sourdough fermentations. J Agric Food Chem 54(11):4078\u0026ndash;4085. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf052504s\u003c/span\u003e\u003cspan address=\"10.1021/jf052504s\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbarra-Molero B, Naganathan AN, Sanchez-Ruiz JM, Mu\u0026ntilde;oz V (2015) Modern analysis of protein folding by differential scanning calorimetry. Methods Enzymol 567:211\u0026ndash;233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/bs.mie.2015.08.027\u003c/span\u003e\u003cspan address=\"10.1016/bs.mie.2015.08.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin C, Geng J, Liu C, Wang Y, Huang J, Xiong YL (2024) Structural changes of flaxseed protein modified by fermentation and the gel properties and swallowing characteristics of its composite system with mung bean starch. LWT 199:116098. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2024.116098\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2024.116098\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaram SAE, Duconseille A, V\u0026eacute;nien A, Ravel C, Chauvet H, Jamme F, R\u0026eacute;fr\u0026eacute;giers M, Astruc T (2025) Change in muscle fibre protein structure following salting process assessed by synchrotron deep UV fluorescence microspectroscopy. Food Chem 471:142801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2025.142801\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2025.142801\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaram SAE, Duconseille A, V\u0026eacute;nien A, Ravel C, Chauvet H, Jamme F, R\u0026eacute;fr\u0026eacute;giers M, Astruc T (2025) Change in muscle fibre protein structure following salting process assessed by synchrotron deep UV fluorescence microspectroscopy. Food Chem 471:142801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2025.142801\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2025.142801\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang J, Xiong YL, Chen J (2010) pH shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions. J Agric Food Chem 58(13):8035\u0026ndash;8042. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf1000178\u003c/span\u003e\u003cspan address=\"10.1021/jf1000178\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan L, Xu H, Huang D (2011) The investigation on states of water in different hydrophilic polymers by DSC and FTIR. J Polym Res 18(4):681\u0026ndash;689. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10965-010-9465-6\u003c/span\u003e\u003cspan address=\"10.1007/s10965-010-9465-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacobs MR, Grace M, Blumlein A, Mcmanus JJ (2019) Differential scanning calorimetry to quantify heat-induced aggregation in concentrated protein solutions. Methods in Molecular Biology 2039: 115\u0026ndash;130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4939-9678-0_9\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-9678-0_9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBates A, Williams KM, Hagerman AE (2025) Protein thermal stability in the undergraduate biochemistry laboratory: Exploring protein thermal stability with yeast alcohol dehydrogenase. Biochem Mol Biol Educ 53(2):226\u0026ndash;234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/bmb.21880\u003c/span\u003e\u003cspan address=\"10.1002/bmb.21880\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurowoju IB, Bhandal KS, Hu J, Carpick B, Kirkitadze M (2017) Differential scanning calorimetry \u0026mdash; A method for assessing the thermal stability and conformation of protein antigen. J Visualized Experiments 121:55262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3791/55262\u003c/span\u003e\u003cspan address=\"10.3791/55262\" 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":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Flaxseed protein, Probiotic fermentation, Thermodynamic properties, Structural reorganization","lastPublishedDoi":"10.21203/rs.3.rs-8463814/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8463814/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlaxseed protein (FP) is a promising plant-based protein source, yet its application in food systems is limited by its inherent structural characteristics. This study systematically investigated the structural and functional evolution of flaxseed protein (FP) during fermentation by \u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e and \u003cem\u003eBifidobacterium lactis\u003c/em\u003e. The fermentation process induced significant time-dependent changes in particle size distribution, shifting from large aggregates (\u0026gt;\u0026thinsp;100 \u0026micro;m) to a homogenized system dominated by 10\u0026ndash;100 \u0026micro;m particles, accompanied by the complete disruption of the native dense microstructure into sub‑50 \u0026micro;m dispersed particles. SDS‑PAGE analysis revealed progressive degradation of high‑molecular‑weight fractions (\u0026gt;\u0026thinsp;100 kDa) and accumulation of low‑molecular‑weight peptides (15\u0026ndash;35 kDa), confirming extensive proteolysis. UV‑spectroscopy indicated a decrease in absorbance at 280 nm and a red‑shift in peak position, suggesting the release of aromatic amino acids and a transition toward disordered conformations. Differential scanning calorimetry demonstrated a marked reduction in denaturation temperature (from 149.27\u0026deg;C to 101.77\u0026deg;C) and an increase in enthalpy change (from 16.77 J/g to 22.80 J/g), reflecting decreased thermal stability and enhanced hydration potential. The results collectively delineate a three‑stage mechanism: initial protease‑driven hydrolysis (0\u0026ndash;4 h), intermediate hydrophobic‑electrostatic recombination (4\u0026ndash;8 h), and final formation of uniform particles (8\u0026ndash;10 h). These structural modifications render fermented FP more suitable for gel‑based food applications by lowering its thermal transition requirements and improving water interaction. This work provides a theoretical foundation for the targeted fermentation design of plant proteins to tailor their techno‑functional properties.\u003c/p\u003e","manuscriptTitle":"Structural and functional evolution of flaxseed protein during probiotic fermentation: Insights into proteolytic degradation, conformational rearrangement, and thermal property","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 17:04:33","doi":"10.21203/rs.3.rs-8463814/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59ae756c-05ac-4241-9dbe-9beaf185660f","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-08T20:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 17:04:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8463814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8463814","identity":"rs-8463814","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}