IntroductionFlaxseed (Linum usitatissimum), an ancient crop from the Linaceae family, is renowned for its health benefits, including regulation of tumor growth, microbial infections, cholesterol, cardiovascular diseases, diabetes, and metabolic syndrome1. These benefits are mainly due to its high content of alpha-linolenic acid, lignans, proteins, phenolic acids, and flavonoids, which enhance the body’s defense against carcinogens and contribute to its protective effects2. Flaxseed is also a rich source of essential nutrients like fat (mainly ALA), protein, fiber, and carbohydrates, making it a valuable addition to the diet1,3.The increasing awareness of the link between diet and health has led to a growing interest in functional foods. Seeds and sprouts from various plants are considered functional foods due to their health benefits, including antioxidant, anticancer, antidiabetic, hypolipidemic, and anti-inflammatory properties. Germination enhances the nutraceutical properties of plants by activating hydrolytic enzymes, which release phenolic compounds from the cell wall into soluble forms4,5,6,7. Additionally, phenolic compounds are produced through metabolic pathways like the pentose phosphate, glycolytic, acetate/malonate, and hydrolytic tannin pathways, contributing to the diversity of these bioactive compounds8,9,10.Beta-glucosidases (EC 3.2.1.21) are important enzymes in many living organisms, including fungi, bacteria, plants, and animals11,12. They are capable of breaking the β-glucosidic bonds of di-, oligosaccharide or other sugar conjugates and play important functions in many biological pathways, including cellulosic biomass degradation and secondary metabolite modification13. During germination, these enzymes cleavage the β-glucosidic bond in phenolic compounds, primarily conjugated to sugar residues bound to the hydroxyl group. This release boosts the level of free polyphenols, which can improve the plant’s health benefits and nutritional value14.Reactive oxygen species (ROS) are critical in seed germination and dormancy regulation. While ROS production is a normal part of plant metabolism, environmental factors can cause excessive ROS production, which must be balanced for proper seed germination and dormancy regulation15. Enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and catalase (CAT), along with non-enzymatic antioxidants like phenolic compounds, proline, and ascorbic acid, help scavenge ROS. CAT and POD convert hydrogen peroxide (H2O2) into water and oxygen, while PPO and POD oxidize phenols to quinone compounds, turning them into dark pigments16. Phenylalanine ammonialyase (PAL) is the first enzyme in phenylpropanoid metabolism, converting aromatic amino acids into trans-cinnamic acid, and its activity is induced during germination17. Studies show that flaxseed germination increases levels of fatty acids, vitamin C, phenolic compounds, and total antioxidant activity while decreasing lipid content during the first week18,19.Although flaxseed is a rich source of bioactive compounds like lignans, phenolic acids, and flavonoids, their bioavailability is often limited by the seed’s structure and antinutritional factors20. While germination is known to improve the nutritional quality of seeds, no study has investigated how phenolic biosynthesis in flaxseed is activated through the action of its endogenous enzymes. Moreover, limited research has connected these biochemical changes to important functional properties such as antioxidant, antibacterial, and glucose-regulating activities. To address this gap, we used a two-level factorial design, which allows multiple factors and their interactions to be studied at the same time, unlike traditional one-factor-at-a-time method21. This approach provides a more complete understanding of how germination and enzymatic activity together enhance phenolic content and its related health benefits.To address these gaps, the current study aimed to enhance the nutritional and functional properties of flaxseeds through controlled germination by:(1) Evaluating the effects of germination on total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity. (2) Investigating the role of specific endogenous enzymes, including β-glucosidase, peroxidase, polyphenol oxidase, and catalase, in modulating these bioactive compounds. (3) Developing a statistical model based on a two-level factorial design, using enzyme activity as input variables to improve total phenolic content (TPC) and total flavonoid content (TFC) biosynthesis during germination. (4) Assessing the resulting biological activities of germinated flaxseed, including antioxidant, antibacterial, and glucose-regulating properties, to support its potential in functional food applications.This study introduces a novel approach by modeling key endogenous enzymes as independent variables in a factorial model to uncover their mechanistic roles and synergistic interactions in phenolic biosynthesis during germination. Unlike the traditional approaches that consider these enzymes as observed outcomes, this strategy provides a clearer understanding of how enzyme activity drives the enhancement of phenolic compounds. It also offers a validated and practical framework for improving the functional quality of flaxseed through targeted germination processes.Materials and methodsSeed germinationThe seeds of Egyptian flax (Linum usitatissimum L.) were collected and identified by the Botany Department at the Agricultural Research Centre (ARC) in Giza, Egypt. The flax seeds (10 g) were surface-sterilized for 5 min with a 0.1% NaClO solution, then rinsed for 15 min with sterile distilled water. The seeds were placed on filter paper in Petri dishes (5 cm in diameter) and incubated at 25 ± 5 °C in the dark. Sprouts were collected daily over 8 days for analysis and stored at − 20 °C8.Preparation of flaxseed extractsTen grams of seeds or sprouts were dried overnight in an oven at 50 °C. The dried material was extracted with 20 ml of 80% methanol (v/v) at 25 °C under agitation (150 rpm) overnight. The extraction mixture was then filtered using Whatman No. 1 filter paper. The filtrate was centrifuged at 4000 rpm for 10 min at 4 °C. After centrifugation, the supernatant was collected, and the solvent was evaporated8.Determination of total phenolic and flavonoid concentrationsThe total phenolic content (TPC) was determined as described by Velioglu et al.22. The reaction mixture contained 50 µl of the methanol extract, 100 µl of Folin–Ciocalteu reagent, and 850 µl of distilled water, and was allowed to stand at room temperature for 5 min. Then, 500 µl of 20% sodium carbonate solution was added, and the mixture was incubated at room temperature for 30 min. The absorbance was measured at 750 nm. Total phenolic content was calculated using a calibration curve prepared with known concentrations of gallic acid and expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (DW).The total flavonoid content (TFC) of the extracts was assessed using a colorimetric method described by Zhishen et al. with slight modifications23. Briefly, 250 µl of the methanol extract was mixed with 1.25 ml of distilled water and 75 µl of 5% NaNO2 solution. After 6 min, 150 µl of 10% AlCl₂ solution was added. One minute later, 0.5 ml of 1 M NaOH and 275 µl of distilled water were added, and the mixture was thoroughly mixed. The absorbance was measured at 510 nm. A standard curve was constructed using catechin in the range of 5–300 mg/kg, and results were expressed as milligrams of catechin equivalents per gram of dry weight (mg CE/g DW).HPLC analysisHPLC analysis was conducted using an Agilent 1100 system with a C18 column and a diode-array detector, following the method of Kim et al.24. A gradient elution of acetonitrile and 2% acetic acid in water was run over 70 min at 1.0 ml/min. The methanol extracts were filtered before being injected (10 µl). Phenolic compounds were identified based on their retention times and UV spectra, which were compared to standard phenolic compounds at wavelengths of 280, 320, and 255 nm.Antioxidant activity (DPPH and ABTS Assays)The antioxidant activity of methanolic flaxseed extracts was assessed using DPPH and ABTS radical scavenging assays25,26. For DPPH: 0.1 mL of extract was mixed with 0.9 mL of 0.1 mM DPPH in methanol, incubated in the dark for 30 min, and absorbance was measured at 517 nm. For ABTS: ABTS⁺ was generated by mixing 7 mM ABTS with 2.45 mM potassium persulfate (1:0.5), incubated for 12–16 h in the dark, then diluted to an absorbance of 0.70 ± 0.02 at 734 nm. A 0.1 mL extract was added to 0.9 mL ABTS solution, and absorbance was read after 1 min.In both assays, \(Scavengingactivity(\% )\, = \left[ {\left( {O.D.{\text{ }}control{\text{ - }}O.D.{\mkern 1mu} sample} \right)} \right]{\mkern 1mu} \, \times 100\)IC50 refers to the concentration of the phenolic extract required to inhibit 50% of DPPH or ABTS free radicals, and was determined by plotting the percentage of radical scavenging activity against varying concentrations of gallic acid equivalents (GAE) in the extract (data not shown).Determination of total antioxidant indexWhile IC50 values are commonly used to measure antioxidant potency, they do not reflect the total antioxidant activity per gram of sample. Therefore, a new calculation was proposed to determine total antioxidant index: Total antioxidant index = Total phenolic content (mg GAE/g DW)/mg IC5010. This method aligns with established recommendations for expressing antioxidant capacity in both concentration- and weight-based formats, and was supported by previous studies8,12,27.Determination of enzymatic activitiesCrude enzyme extractionOne gram of flax seeds or sprouted seeds was homogenized with 20 mM Tris–HCl buffer (pH 7.2) using a glass homogenizer. The resulting homogenate was centrifuged at 13,500 × g for 10 min at 4 °C. The supernatant obtained was considered the crude extract and was stored at − 20 °C for subsequent analysis8,10.Assay of PAL activityThe activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) was assessed following the method outlined by Goldson et al.28. The reaction mixture consisted of 0.1 ml of crude enzyme extract, 40 mM phenylalanine, and 20 mM Tris–HCl (pH 8.8). The mixture was incubated at 37 °C for 30 min. To terminate the reaction, 200 µl of 2% trichloroacetic acid (TCA) was added after incubation, and the samples were then centrifuged at 13,000 × g for 15 min. The absorbance at 290 nm was measured to quantify the amount of trans-cinnamic acid produced, which was determined using a standard curve.β-GL activity assayβ-Glucosidase (β-GL) activity was measured as described by Gunata et al.29. The enzyme activity was assayed by incubating 0.9 mM p-nitrophenyl-β-D-glucopyranoside in 20 mM acetate buffer (pH 5.5) with 0.1 ml of the enzyme solution at 37 °C for 20 min. The reaction was terminated by adding 0.6 ml of 1 M sodium carbonate. The amount of p-nitrophenol released was quantified by measuring the absorbance at 405 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol of p-nitrophenol per minute.POX assayPeroxidase (POX) activity was measured according to Miranda et al.30. The reaction mixture consisted of 1 mL containing 8 mM H₂O₂, 40 mM guaiacol, 20 mM sodium acetate buffer (pH 5.5), and 0.1 mL of crude extract. The variation in absorbance at 470 nm, resulting from the oxidation of guaiacol, was monitored for 1 min using a spectrophotometer at ambient temperature. One unit of peroxidase activity is defined as the amount of enzyme that causes an increase of 1.0 OD per minute under the specified assay conditions. The activity is expressed in U/g seeds.CAT assayCatalase (CAT) activity was quantified using the method described by Bergmeyer31. A substrate solution consisting of 25 mM H₂O₂ in 75 mM sodium phosphate buffer (pH 7.0) was prepared, and 1 ml of this solution was combined with 0.1 ml of crude extract. The reduction in absorbance at 240 nm was monitored for 1 min using a spectrophotometer at ambient temperature. One unit of enzyme activity is defined as the amount of enzyme that induces a change of 0.1 absorbance units per minute under the specified assay conditions. The activity is expressed in U/g seeds.PPO assayPolyphenol oxidase (PPO) activity was measured according to Jiang et al.32. A crude enzyme extract (0.1 ml) was quickly added to a 0.02 M catechol solution, prepared in a 0.01 M sodium phosphate buffer (pH 6.8). The change in absorbance was measured at 400 nm and room temperature. One unit of PPO activity is defined as the amount of enzyme that causes an increase of 0.1 in absorbance (O.D.) per minute under the specified assay conditions. The activity is expressed in U/g seeds.Anti-bacterial propertiesThe antibacterial activity of phenolic extracts from flaxseeds and 5-day sprouts was evaluated against Escherichia coli and Staphylococcus aureus using the agar well diffusion method33. Bacterial suspensions (10⁸ CFU/ml) were spread on Mueller–Hinton agar, and 50 µg GAE of each extract was added to wells, followed by incubation at 37 ± 1 °C for 18 h. Inhibition zones were measured to assess activity, with gentamicin as a control. The minimum inhibitory concentration (MIC) was determined via agar dilution, identifying the lowest extract concentration that inhibited visible bacterial growth after 18 h at 37 ± 1 °C.Anti-hyperglycemic propertiesThe in vitro anti-hyperglycemic potential of phenolic extracts from flaxseeds and 5-day sprouts was assessed through α-amylase and α-glucosidase inhibition assays, following the methods of Liu et al.34 and Zhang et al.35, respectively. For α-amylase inhibition, a reaction mixture containing 5 U of pancreatic α-amylase, sodium phosphate buffer (pH 7.2), and either the extract or acarbose was incubated at 37 °C, followed by starch addition, and absorbance was measured at 540 nm after color development with dinitro-salicylic reagent. α-Glucosidase inhibition was measured using one unit of enzyme with p-nitrophenyl-α-glucopyranoside as substrate in a buffer at pH 6.8, with absorbance read at 405 nm. In both assays, the percentage of enzyme inhibition was calculated based on the decrease in optical density, and the IC50 value represented the extract concentration required to inhibit 50% of the enzyme activity.Experimental designTo investigate the factors influencing the release of total phenolic content (TPC) and total flavonoid content (TFC) during flaxseed germination, a two-level factorial design was employed using Design-Expert® Software Version 11 (Stat-Ease Inc., Minneapolis, MN, USA). This design was developed based on findings from preliminary single-variable experiments, which helped identify the most influential enzymatic and germination-related factors. Six independent variables were selected based on their known roles in phenolic biosynthesis and metabolism: germination day (GD), β-glucosidase (β-GL), phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO), and catalase (CAT). Each factor was evaluated at two levels (low and high) to assess its effect on TPC and TFC production. The experimental design consisted of 14 runs, including 7 factorial combinations replicated in two batches. Each treatment was performed in triplicate to ensure statistical reliability. The data were analyzed using factorial modeling to evaluate both the main effects and interactions between the variables. An empirical model was generated to describe the relationship between the input variables and the measured responses (TPC and TFC), allowing for the identification of significant and synergistic effects.An empirical relationship between the input parameters and the output responses was established using regression analysis, as shown below:TPC equationsIn terms of actual factors$$\begin{gathered} {\text{TPC }} = {\text{ }} + {\text{6}}.{\text{424 }} - {\text{ 2}}.{\text{315 }} \times \,{\text{GD }} + {\text{ }}0.{\text{348}}\, \times \,\beta - {\text{GL }} - {\text{ }}0.{\text{882}}\,\, \times \,{\text{PAL }} + 0.0{\text{2}}0\, \hfill \\ \times {\text{POX }} + {\text{ }}0.0{\text{22}} \times {\text{PPO }} + {\text{ }}0.0{\text{4}}0{\text{2}} \times {\text{CAT }} + {\text{ }}0.{\text{136}} \times {\text{ }}\left( {{\text{GD }} \times {\text{ }}\beta - {\text{GL}}} \right){\text{ }} \hfill \\ - {\text{ }}0.00{\text{4}}\beta - {\text{GL}} \times {\text{PPO}} + {\text{ }}0.00{\text{4}} \times {\text{ }}\left( {{\text{PAL }} \times {\text{ PPO}}} \right) \hfill \\ \end{gathered}$$In terms of coded factors$$\begin{gathered} {\text{TPC }} = {\text{ }} + {\text{1}}0.{\text{18 }} + {\text{5}}.{\text{51 A }} + {\text{ 2}}.{\text{35 B }} - {\text{1}}.{\text{6}}0{\text{ C }} + {\text{2}}.{\text{38 D }} - {\text{2}}.{\text{67 E }} \hfill \\ + {\text{1}}.{\text{54 F }} + {\text{4}}.{\text{41 AB }} - {\text{5}}.{\text{83 BE }} + {\text{5}}.{\text{81 CE}} \hfill \\ \end{gathered}$$TFC equationsIn terms of actual factors$$\begin{gathered} {\text{TFC }} = {\text{ }} - {\text{1}}.0{\text{11 }} + {\text{ }}0.{\text{182}} \times {\text{GD }} - {\text{ }}0.0{\text{27}} \times \beta - {\text{GL }} - {\text{ }}0.0{\text{29}} \times {\text{PAL }} - {\text{ }}0.0{\text{165}} \times {\text{POX }} \hfill \\ - {\text{ }}0.0{\text{38}} \times {\text{PPO }} + {\text{ }}0.0{\text{45}} \times {\text{CAT }} + {\text{ }}0.00{\text{4}} \times \,\,\left( {{\text{GD }} \times {\text{ POX}}} \right){\text{ }} + {\text{ }}0.00{\text{1}} \times \hfill \\ {\text{ }}\left( {\beta - {\text{GL }} \times {\text{ PPO}}} \right){\text{ }} - {\text{ }}0.000{\text{72}} \times \left( {\beta - {\text{GL }} \times {\text{ CAT}}} \right) \hfill \\ \end{gathered}$$In terms of coded factors$$\begin{gathered} {\text{TFC }} = {\text{ }} + {\text{1}}.0{\text{7 }} + {\text{ 2}}.{\text{84 A }} + 0.{\text{9294 B }} - 0.{\text{2685 C }} + 0.{\text{5791 D }} - \hfill \\ 0.{\text{4177 E }} + 0.{\text{9388 F }} + {\text{1}}.{\text{97 AD }} + {\text{1}}.{\text{65 BE }} - 0.{\text{2566 BF}} \hfill \\ \end{gathered}$$These models were used to identify significant contributors and interactions in the biosynthesis of phenolic and flavonoid compounds during germination.Statistical analysisThe data were analyzed using a one-way ANOVA, followed by Tukey’s post hoc test and correlation analysis, all performed with GraphPad Prism 5 software. Results are presented as means ± standard deviation (n = 4), with statistical significance at P