Fresh ground beef (80% lean, 20% fat) was obtained from a local meat processing facility in Taif, Saudi Arabia. Chia seeds (Salvia hispanica L.) were purchased from Al-Dawaa Pharmacy (Taif, Saudi Arabia). Seeds were manually cleaned to remove foreign materials and stored in airtight containers at ambient temperature until further use. All analytical-grade reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), including thiobarbituric acid (TBA; cat. no. T5500), trichloroacetic acid (TCA; cat. no. T6399), sodium chloride (NaCl; cat. no. S9888), butylated hydroxytoluene (BHT; cat. no. W218405), and malondialdehyde (MDA) standard for TBARS analysis (cat. no. MDA325). Microbiological media were obtained from Merck (Darmstadt, Germany), including plate count agar (PCA; cat. no. 105463), MacConkey agar (cat. no. 105465), and potato dextrose agar (PDA; cat. no. 110130). Distilled water was used for the preparation of all solutions and for equipment cleaning.

Chia seeds (Salvia hispanica) were dried in a hot-air oven at 40 $\pm$ 2 °C for 12 h to reduce moisture content. The dried seeds were ground into a fine powder using a laboratory grinder (Kenwood Multi Mill, Model BL335, cat. no. N/A, Kenwood Appliances, Taif, Saudi Arabia) and sieved through a 60-mesh screen to ensure uniform particle size. For extraction, 50 g of chia seed powder was mixed with 500 mL of distilled water (1:10 w/v) and stirred at 45 °C for 2 h using a magnetic stirrer (MS300HS, cat. no. 88880013, Thermo Fisher Scientific, Waltham, MA, USA). The extraction procedure was selected based on food-processing feasibility and previously reported aqueous extraction methods for plant phenolics to ensure practical relevance for potential industrial application. The mixture was cooled to room temperature and filtered through Whatman No. 1 filter paper (cat. no. 1001-110, Cytiva/Whatman, Marlborough, MA, USA). The filtrate was freeze-dried using a bench-top freeze dryer (FreeZone 2.5 L, cat. no. 7670520, Labconco Corporation, Kansas City, MO, USA) to obtain chia seed extract powder, which was stored in airtight containers at 4 °C until further use in burger formulation.

The proximate and bioactive composition of chia seed (Salvia hispanica L.) powder was analyzed prior to extract preparation using Association of Official Analytical Collaboration (AOAC) methods [18] and complementary instrumental techniques. All analyses were performed in triplicate, and results were expressed as mean $\pm$ standard deviation.

The vitamin content of chia seed (Salvia hispanica) powder was quantified using standardized methods to assess its nutritional contribution as a functional ingredient.

The antioxidant capacity of chia seed (Salvia hispanica) powder extract was evaluated using three complementary in vitro assays: ABTS⁺ radical scavenging activity, DPPH radical scavenging activity, and ferric reducing antioxidant power (FRAP). All measurements were performed in triplicate, and results were expressed as µmol Trolox equivalents (TE) per gram of extract.

The tannic acid content of chia seed (Salvia hispanica) powder extract was determined spectrophotometrically using the Folin–Denis method, as described by [18, 19] with minor modifications. An aliquot of 1 mL of chia seed extract (1 mg/mL) was mixed with 1 mL of Folin–Denis reagent (cat. no. F9252, Sigma-Aldrich, St. Louis, MO, USA), followed by 2 mL of saturated sodium carbonate solution (cat. no. S7795, Sigma-Aldrich, St. Louis, MO, USA). The mixture was diluted to 10 mL with distilled water and incubated at room temperature for 30 min in the dark. The absorbance of the resulting blue color was measured at 760 nm using a UV–Vis spectrophotometer (UV-1800, cat. no. N/A, Shimadzu Corporation, Kyoto, Japan). Tannic acid (cat. no. T0377, Sigma-Aldrich, St. Louis, MO, USA) was used as the standard, and a calibration curve was prepared using concentrations ranging from 0 to 100 µg/mL. The tannic acid content was calculated from the standard curve and expressed as milligrams of tannic acid equivalents per gram of extract (mg TAE/g extract).

The physicochemical, microbiological, and sensory properties of burger samples treated with chia seed extract were evaluated at regular intervals (0, 3, 6, 9, and 12 days) during refrigerated storage at 4 $\pm$ 1 °C, according to methods described below.

All experimental data were analyzed using SPSS software (version 25.0; IBM Corp., Armonk, NY, USA). Results are expressed as mean $\pm$ standard deviation (SD) of three independent replicates (n = 3). One-way analysis of variance (ANOVA) was performed to evaluate differences among treatments and storage periods. When significant effects (p 0.05) were detected, Duncan’s multiple range test was applied for post hoc pairwise comparisons. Sensory evaluation data were analyzed using the non-parametric Kruskal–Wallis test, with the Mann–Whitney U test conducted for pairwise comparisons where appropriate. Graphs and figures were generated using GraphPad Prism (version 9; GraphPad Software Inc., San Diego, CA, USA). A probability level of p 0.05 was considered statistically significant for all tests.

The proximate composition of chia seed powder demonstrates its exceptional nutritional value, supporting its use in functional food systems (Table 2). Moisture content was low (5.84 $\pm$ 0.12%), consistent with effective drying protocols, which enhance shelf-life and protect against microbial and enzymatic deterioration. Protein content was 20.31 $\pm$ 0.24%, within the reported range for chia (16–23%), and rich in essential amino acids such as arginine and leucine, contributing to cardiovascular health, tissue repair, and muscle metabolism [20, 22]. Lipid content was high (31.66 $\pm$ 0.45%), predominantly polyunsaturated fatty acids (PUFAs), with $\alpha$-linolenic acid (ALA, $\omega$-3) comprising 62.3% of total fatty acids, providing a plant-based alternative for omega-3 fortification [23, 24]. Dietary fiber (23.40 $\pm$ 0.33%) included soluble (7.12 $\pm$ 0.21%) and insoluble (16.28 $\pm$ 0.26%) fractions, supporting gastrointestinal health, glycemic regulation, and satiety [24, 25]. Essential minerals were abundant, particularly calcium (590.4 $\pm$ 10.6 mg/100 g), magnesium (335.7 $\pm$ 8.4 mg/100 g), potassium (722.1 $\pm$ 12.3 mg/100 g), and phosphorus (744.5 $\pm$ 9.8 mg/100 g), promoting bone, neuromuscular, and metabolic functions [25].

Chia seeds were also rich in bioactive compounds: total phenolic content (TPC) was 38.72 $\pm$ 1.34 mg GAE/g, flavonoids 22.18 $\pm$ 0.97 mg QE/g, and tannins 12.94 $\pm$ 0.88 mg TAE/g, conferring strong antioxidant potential [26, 27]. Antioxidant assays confirmed this activity: DPPH radical scavenging was 75.61 $\pm$ 1.22%, ABTS⁺ inhibition 81.47 $\pm$ 1.08%, and ferric reducing antioxidant power (FRAP) 62.53 $\pm$ 1.17 µmol TE/g. HPLC analysis revealed abundant phenolics such as gallic acid (8.37 mg/g), caffeic acid (4.15 mg/g), and ferulic acid (3.89 mg/g), compounds known for anti-inflammatory, antimicrobial, and anti-aging effects [27, 28].

The high bioactivity of chia seed extract, demonstrated through multiple assays and a diverse phenolic profile, highlights its dual functionality: it can retard lipid oxidation and enhance oxidative stability in lipid-rich foods, including ground beef, while contributing nutritional and health-promoting benefits. Incorporation of chia extract into meat products thus represents a promising natural alternative to synthetic antioxidants, aligning with consumer demand for clean-label, functional ingredients [29].

Regarding vitamin composition, chia seed powder exhibited a diverse profile, providing both water- and fat-soluble vitamins. Vitamin C content was 7.28 $\pm$ 0.18 mg/100 g, supporting its contribution to immune defense and collagen synthesis. Among B-complex vitamins, niacin (5.63 mg/100 g) was particularly abundant, complementing energy metabolism and neurological function. Additionally, the seeds were a rich source of vitamin E (12.84 $\pm$ 0.37 mg/100 g), a lipid-soluble antioxidant that protects cellular membranes and unsaturated fatty acids from peroxidation. The coexistence of vitamin C and E suggests synergistic antioxidant interactions, further enhancing chia’s functional role in stabilizing food systems. The compositional richness of chia seed powder—characterized by its high omega-3 fatty acids, substantial dietary fiber, mineral density, and abundant polyphenolic compounds—confirms its value as a multifunctional ingredient. Its strong antioxidant activity, combined with essential micronutrients, makes it an ideal candidate for incorporation into health-oriented food formulations. Potential applications span bakery, dairy, and particularly meat products, where chia can enhance shelf-life, improve nutritional labeling, and increase consumer appeal. Importantly, its ability to replace synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) aligns with clean-label trends and regulatory shifts toward natural preservation strategies.

High-performance liquid chromatography (HPLC) was employed to identify and quantify the major phenolic compounds present in chia seed (Salvia hispanica L.) extract, providing insights into its antioxidant potential and suitability as a functional food ingredient (Fig. 1; Tables 3,4). The analysis was performed using a Shimadzu LC-20AT HPLC system equipped with a diode array detector (DAD; Shimadzu Corp., Kyoto, Japan), allowing precise profiling of phenolic constituents based on retention time and UV-visible spectra. The chia extract was dissolved in HPLC-grade methanol, filtered through a 0.45 µm PTFE syringe filter, and stored at 4 °C until analysis. Separation was achieved on a C18 reverse-phase column (250 mm $\times$ 4.6 mm, 5 µm particle size), providing optimal resolution for plant-derived phenolics. Compound identification was based on comparison with authentic standards, including gallic acid, caffeic acid, chlorogenic acid, ferulic acid, rutin, and quercetin. Quantification was performed using external standard calibration curves, and all determinations were carried out in triplicate. Results were expressed as mg/g dry extract (mean $\pm$ standard deviation). The resulting chromatogram (Fig. 1) exhibited a complex phytochemical profile with 48 distinct peaks. Early-eluting peaks (e.g., peaks 4, 6, and 10) corresponded to smaller, more polar phenolic acids, whereas the most intense peaks were observed between 22 and 24 minutes, representing chlorogenic acid, ferulic acid, quercetin, and related flavonoids. This elution pattern aligns well with previously reported chia seed phenolic profiles [29, 30].

Fig. 1.

Base peak chromatogram of chia seed (Salvia hispanica L.) extract obtained by high-performance liquid chromatography with diode array detection (HPLC-DAD). The chromatogram exhibits a complex phenolic profile comprising 48 distinct peaks eluting between 2.1 and 26.0 minutes. Early-eluting peaks (2–10 min), including gallic acid, protocatechuic acid, and p-hydroxybenzoic acid, correspond to low-molecular-weight, highly polar hydroxybenzoic acids, while later peaks represent less polar phenolics, including ferulic acid, rutin, and quercetin. The most intense peaks were observed between 22 and 24 minutes, corresponding to high-intensity flavonoid compounds (see Table 4). Peaks 13 and 15 represent to minor early-eluting traces of rutin and quercetin; however, their major quantitative signals were recorded at 32.48 and 39.72 min, respectively. UV absorbance was monitored at 280 nm and 320 nm for optimal detection of phenolic acids and flavonoids. The chromatographic pattern, consistent with Table 3, reflects the diverse composition of hydroxybenzoic, hydroxycinnamic, and flavonol derivatives, which collectively contribute to the potent antioxidant potential of chia seed extract.

Quantitative analysis (Table 4) identified gallic acid (8.37 mg/g), caffeic acid (4.15 mg/g), and ferulic acid (3.89 mg/g) as the predominant phenolic compounds, accompanied by appreciable levels of chlorogenic acid (2.91 mg/g), rutin (2.46 mg/g), and quercetin (1.74 mg/g). Overall, hydroxybenzoic acids (e.g., gallic acid) and flavonols (e.g., quercetin, rutin) were the most abundant phenolic classes. These compounds are recognized for their potent antioxidant, anti-inflammatory, and antimicrobial properties. From a functional perspective, gallic and caffeic acids are known inhibitors of lipid peroxidation, while flavonols such as quercetin and rutin contribute cardiovascular, neuroprotective, and anti-carcinogenic effects. The phenolic composition observed in chia seed extract, therefore, underscores its potential as a natural antioxidant source capable of enhancing lipid stability, delaying rancidity, and improving the nutritional and functional quality of processed meat products. These findings highlight chia seed extract’s promise as a clean-label alternative to synthetic antioxidants, supporting current trends toward natural preservation strategies in the food industry. High-performance liquid chromatography with diode array detection (HPLC-DAD) was employed to characterize and quantify the major phenolic constituents of chia seed (Salvia hispanica L.) extract, providing insight into its antioxidant potential and suitability as a natural functional ingredient. The chromatographic analysis revealed a rich and complex phenolic profile, as illustrated in Fig. 1, comprising a total of 48 distinct peaks across the retention range of 2.1–26.0 minutes. The identified compounds were tentatively assigned based on retention times, UV spectral characteristics, and comparison with authentic standards and previously reported chromatographic data (Table 3).

Early-eluting peaks, particularly those appearing between 2 and 10 minutes, corresponded predominantly to low-molecular-weight, highly polar phenolic acids. These included gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, chlorogenic acid, and ferulic acid, among others. Such compounds belong primarily to the hydroxybenzoic and hydroxycinnamic acid classes, which are well known for their strong antioxidant and radical-scavenging activity. Mid-to-late eluting peaks, detected between 15 and 26 minutes, represented larger, less polar compounds such as flavonols and flavones, including myricetin, quercetin, kaempferol, luteolin, and apigenin. The presence of both phenolic acids and flavonoids underscores the chemical diversity of chia seed extract, reflecting its multifunctional bioactivity potential.

Notably, peaks 13 and 15 corresponded to early-eluting traces of rutin and quercetin; however, their major quantitative signals were observed at 32.48 min and 39.72 min, respectively. These later peaks were used for quantification and are presented in Table 4. The chromatogram thus exhibits a bimodal pattern typical of plant extracts, where early peaks indicate polar phenolic acids and later peaks represent high-molecular-weight flavonoids. UV absorbance was monitored at 280 nm and 320 nm, which are optimal wavelengths for detecting phenolic acids and flavonoids, respectively.

Quantitative analysis (Table 4) revealed that gallic acid (8.37 $\pm$ 0.24 mg/g dry extract) was the predominant phenolic compound in chia seed extract. This hydroxybenzoic acid is widely recognized for its potent antioxidant, antimicrobial, and antimutagenic properties, and its abundance correlates with the high total phenolic content (TPC) and strong radical scavenging activity observed in DPPH and ABTS assays. Caffeic acid (4.15 $\pm$ 0.17 mg/g) and ferulic acid (3.89 $\pm$ 0.21 mg/g) were also detected in substantial quantities. These hydroxycinnamic acids are effective in neutralizing free radicals, chelating transition metals, and inhibiting lipid peroxidation, which makes them particularly valuable for extending the shelf-life of lipid-rich foods, including meat products [31, 32, 33].

Chlorogenic acid (2.91 $\pm$ 0.15 mg/g) was another significant hydroxycinnamic derivative identified, contributing synergistically to antioxidant capacity. It is additionally associated with antidiabetic, metabolic, and neuroprotective effects [34], further enhancing the nutraceutical potential of chia extract. Among the flavonoids, rutin (2.46 $\pm$ 0.12 mg/g) and quercetin (1.74 $\pm$ 0.11 mg/g) were confirmed as key constituents. Both belong to the flavonol class and are known for their anti-inflammatory, antihypertensive, and cardioprotective actions [35, 36]. Notably, rutin—a glycosylated flavonol—exhibits high thermal stability, maintaining its antioxidant capacity during thermal processing, making it suitable for incorporation into cooked or baked products.

The coexistence of hydroxycinnamic acids and flavonols in chia seed extract highlights its multifunctional role in food systems. These compounds not only enhance the extract’s antioxidant activity but also contribute to technological benefits, including improved lipid stability, color retention, and moisture preservation. In meat formulations, for instance, chia seed extract can significantly suppress oxidative deterioration, extend product freshness, and reduce reliance on synthetic antioxidants such as BHT and BHA. Functionally, the extract offers dual benefits—enhancing nutritional value through bioactive phenolics while simultaneously improving product stability by mitigating oxidative processes. Additionally, the phenolic matrix contributes to flavor and color stabilization, both crucial for maintaining consumer acceptability during storage.

The chromatographic and quantitative findings (Tables 3,4) demonstrate that chia seed extract is a potent source of both hydroxybenzoic and hydroxycinnamic acids, complemented by biologically active flavonols. The predominance of gallic acid, together with significant levels of caffeic, ferulic, and chlorogenic acids, ensures robust radical-scavenging activity, while rutin and quercetin further enhance biological efficacy and processing stability. This chemical composition confirms chia seed extract as a natural, clean-label antioxidant with high potential for application in oxidation-sensitive foods, particularly processed meats. Such findings substantiate the extract’s dual role as a nutraceutical and natural preservative, supporting the development of healthier and more sustainable food preservation strategies in response to increasing consumer demand for synthetic additive-free products.

This analytical approach enabled a comprehensive characterization of the bioactive composition of chia seed (Salvia hispanica L.) extract, forming the foundation for understanding its antioxidant efficacy and potential applications in food systems. The identified phenolic compounds (Table 4) emphasize chia’s dual functionality as both a nutraceutical and a natural preservative, supporting its incorporation into functional and shelf-stable food formulations. Among the detected compounds, gallic acid emerged as the most abundant constituent (8.37 $\pm$ 0.24 mg/g dry extract). As a hydroxybenzoic acid, gallic acid is renowned for its strong antioxidant, antimicrobial, and antimutagenic properties. Its predominance within the extract corresponds with the high total phenolic content (TPC) and the elevated radical-scavenging capacity observed in antioxidant assays. Substantial levels of caffeic acid (4.15 $\pm$ 0.17 mg/g) and ferulic acid (3.89 $\pm$ 0.21 mg/g) were also detected. These hydroxycinnamic acids are extensively documented for their ability to neutralize free radicals, chelate pro-oxidant metals, and inhibit lipid peroxidation, thereby contributing significantly to the extension of meat product shelf-life by mitigating oxidative rancidity [32, 33]. Chlorogenic acid (2.91 $\pm$ 0.15 mg/g), another abundant hydroxycinnamic derivative, exhibited synergistic antioxidant effects alongside gallic and caffeic acids. Beyond its antioxidative capacity, chlorogenic acid has been linked to metabolic regulation, antidiabetic, and neuroprotective properties [34], enhancing the extract’s value as a functional bioactive ingredient in health-oriented food applications. Among the flavonoids identified, rutin (2.46 $\pm$ 0.12 mg/g) and quercetin (1.74 $\pm$ 0.11 mg/g) were particularly prominent. These flavonols are associated with anti-inflammatory, antihypertensive, and cardioprotective effects [35, 36]. Rutin, a glycosylated form of quercetin, is noteworthy for its superior stability under heat, maintaining antioxidant integrity during thermal processing—a property that supports its incorporation into cooked or baked food matrices. Together, rutin and quercetin substantially enhance the extract’s biological potency and processing resilience.

The co-occurrence of hydroxycinnamic acids and flavonols in chia seed extract underscores its multifunctionality in food systems. These compounds not only provide potent antioxidant protection but also influence technological attributes such as lipid stability, moisture retention, and color preservation. Their presence contributes to the improvement of organoleptic properties and storage stability in formulated products. In meat-based systems, for instance, the addition of chia extract has the potential to suppress oxidative degradation, prolong product freshness, and reduce the dependence on synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). From a formulation perspective, chia extract fulfills a dual role—enhancing nutritional quality through its diverse bioactive compounds, while simultaneously improving oxidative stability through its phenolic matrix. This duality extends to functional outcomes such as flavor protection, color stabilization, and maintenance of sensory quality throughout storage. The synergistic contribution of gallic, caffeic, ferulic, and chlorogenic acids ensures robust radical-scavenging performance, while rutin and quercetin further amplify biological benefits and thermal resilience.

Overall, the diversity and abundance of phenolic acids and flavonoids identified in chia seed extract confirm its potential as a multifunctional, clean-label additive for oxidation-sensitive foods. The compositional profile (Tables 3,4) illustrates a balance between polar phenolic acids and less polar flavonoids, reflecting a phytochemical spectrum capable of broad antioxidant coverage. These findings substantiate chia seed extract’s application as a natural preservative and nutraceutical, capable of enhancing both the nutritional and technological quality of food products. In particular, its efficacy in stabilizing lipid systems and suppressing oxidative rancidity positions chia extract as a promising alternative to synthetic antioxidants, aligning with current industry trends favoring healthier and more sustainable preservation strategies.

The proximate composition of beef burger samples enriched with chia seed (Salvia hispanica L.) extract was evaluated on day one of refrigerated storage to determine its influence on product nutritional quality. Moisture content ranged from 69.60 to 71.16 g/100 g across all formulations, with no significant differences observed among treatments (p $>$ 0.05). Although chia-enriched groups (T1–T3) exhibited marginally lower moisture levels compared to controls, the reduction was not substantial. This slight decrease may be related to the hydrophilic polysaccharides naturally present in chia seeds, which can bind water and stabilize the meat matrix, thereby minimizing free water release [36]. Protein content showed a modest increase in the chia-supplemented formulations relative to the control samples. Treatments T2 and T3 recorded values exceeding 16 g/100 g, suggesting that chia extract can contribute incrementally to the protein fraction of the product. This improvement is consistent with the high-quality protein profile of chia seeds, which contain 20–23% protein enriched with essential amino acids [37]. The incorporation of chia seed extract, therefore, not only preserves the proximate composition of beef burgers but also offers an avenue for nutritional enhancement, particularly in terms of protein contribution. These findings are presented in Table 5 and Fig. 2.

Fig. 2.

Proximate composition of beef burgers prepared with chia seed extracts during cold moisture content decreased gradually in all beef burger treatments during refrigerated storage, a typical trend attributed to water migration and evaporation. (A) Moisture content: Shows changes in moisture percentage of control and chia-supplemented beef burgers (T1–T3) during cold storage, reflecting water migration and retention behavior. (B) Protein content: Illustrates the effect of chia seed extract incorporation and storage time on protein levels of beef burgers. (C) Fat content: Demonstrates variations in fat percentage among treatments during refrigerated storage. (D) Ash content: Represents changes in total mineral (ash) content of beef burgers as affected by chia seed extract concentration and storage duration. (E) Carbohydrate content: Displays the influence of chia seed extract addition on calculated carbohydrate content during storage. Chia-supplemented samples (T1–T3) initially exhibited slightly lower moisture values, likely due to the water-binding and gel-forming properties of chia polysaccharides. By day 12, however, T3 (1.5% chia extract) demonstrated superior moisture retention compared with the other chia treatments, suggesting that higher chia incorporation may stabilize water distribution within the meat matrix.

A statistically significant increase in lipid content was observed in treatment T3 (10.98 $\pm$ 0.70 g/100 g) compared with both control and SET groups (p $\le$ 0.05). This elevation is likely attributable to the intrinsic lipid fraction of chia seeds, which naturally contain 30–35% oil, particularly rich in omega-3 fatty acids such as $\alpha$-linolenic acid [38].

Thus, higher levels of chia extract incorporation not only increase total lipid content but may also enhance the fatty acid profile of the product, contributing to its nutritional value. Ash content remained consistent across all formulations, indicating that chia addition did not markedly alter total mineral content. Minor increases in carbohydrate levels were noted in treatments T1 and T2, though these changes were not statistically significant (p $>$ 0.05). Such variations may be related to the presence of dietary fiber or residual polysaccharides derived from the chia extract matrix. The pH values of all samples ranged between 6.00 and 6.11, with no significant differences detected (p $>$ 0.05). This stability suggests that chia seed extract does not interfere with the initial acidity of the meat matrix, a favorable outcome for maintaining microbial stability during storage. Overall, these findings confirm that chia seed extract can be successfully incorporated into beef burger formulations without negatively impacting proximate composition. On the contrary, its inclusion may enhance the protein fraction and lipid quality while also offering secondary functional benefits—such as improved antioxidant stability, enhanced water-holding capacity, and prolonged shelf-life—which are examined in subsequent storage studies.

Lipid content also declined slowly across all groups throughout storage, most likely as a result of oxidative degradation. T3, which began with the highest lipid content, retained more fat than other treatments during storage. This observation suggests a lipid stabilization of chia seed extract, attributable to its richness in polyphenols, flavonoids, and tocopherols that may inhibit lipid oxidation. Table 6 (Ref. [39]) presents the changes in cooking yield and shrinkage percentage of beef burgers fortified with chia seed extracts (CSEs) over 15 days of refrigerated storage. Cooking yield, an essential technological parameter, reflects the ability of a product to retain water and fat during heat treatment, while shrinkage corresponds to the dimensional and weight losses that occur during cooking. Both parameters are critical indicators of product quality, consumer acceptability, and industrial profitability [40, 41]. At day 0, the incorporation of chia seed extracts significantly improved (p $\le$ 0.05) the cooking yield compared with both the control and SET (synthetic extract-treated) samples. Among treatments, T2 (89.45%) and T3 (87.47%) displayed the highest yields, surpassing the control group (83.61%). This initial enhancement can be attributed to the high mucilage and soluble dietary fiber content of chia seeds, which develop a viscous, gel-like network during mixing and cooking. These hydrocolloidal structures effectively bind and entrap free water and lipids within the meat matrix, minimizing drip and fat loss during heat treatment [35, 42].

By day 5, a gradual reduction in cooking yield was observed across all samples, likely associated with early protein denaturation and oxidative interactions that reduce the water-holding capacity of myofibrillar proteins. Nonetheless, CSE-treated burgers maintained significantly higher yields compared to the control, confirming the stabilizing role of chia bioactives during short-term storage. The yield values for T2 (88.26%) and T1 (86.63%) remained particularly high, indicating that chia extract effectively mitigates the moisture and fat loss commonly seen during initial storage stages. As storage progressed to day 10, all samples showed a marked decline in cooking yield, with the control and SET dropping to 73.80% and 74.92%, respectively. This reduction can be attributed to protein oxidation, structural weakening, and increased water mobility within the meat matrix. However, chia-supplemented burgers demonstrated a significantly improved performance. Notably, T3 (84.34%) retained substantially higher yield compared with all other treatments (p $\le$ 0.05), suggesting that the antioxidant phenolics and polysaccharides present in chia extracts play a key role in stabilizing protein–water interactions and maintaining emulsion integrity under storage stress.

Interestingly, by day 15, a notable increase in cooking yield was recorded for all chia-supplemented samples, with T3 reaching the highest yield (91.27%), followed by T2 (87.11%), both markedly outperforming the control (83.89%) and SET (82.87%). This apparent recovery may be explained by the water-binding and film-forming capacity of chia mucilage, which may continue to reinforce the meat matrix structure during cold storage. The chia extracts’ polyphenolic components, particularly gallic, caffeic, and ferulic acids, may also inhibit oxidative degradation of muscle proteins and lipids, thereby preserving the physicochemical conditions necessary for optimal water retention [43, 44]. These interactions likely prevent excessive protein cross-linking and preserve a more hydrated network, contributing to higher yields at the final storage stage.

Overall, the data indicate that chia seed extract supplementation significantly enhances the technological functionality of meat systems by improving cooking yield. The improvement is both immediate (day 0)—due to hydrocolloid formation—and sustained (up to day 15)—through antioxidant protection and protein–polysaccharide interactions. Treatments containing higher concentrations of chia extract (particularly T3) consistently achieved superior performance throughout storage, underscoring a dose-dependent relationship between chia content and yield stability.

In contrast, the control and SET samples exhibited greater fluctuations and overall lower yields, suggesting that chia’s combination of hydrophilic fibers and bioactive compounds provides a more comprehensive stabilizing effect than conventional synthetic antioxidants. The ability of chia components to form viscous networks and retain moisture also contributes to better product juiciness, mouthfeel, and consumer acceptability. From a mechanistic perspective, chia’s hydrocolloids act as natural water binders, reducing drip loss during cooking, while the antioxidants and phenolic acids (e.g., gallic, ferulic, and chlorogenic acids) counteract oxidative stress, maintaining protein functionality. This synergistic behavior enhances the product’s structural integrity, yielding higher cooking recovery rates and lower post-cooking deformation. Collectively, the results demonstrate that chia seed extract supplementation serves a dual functional role in meat systems:

(1) Technological: by improving cooking yield and preserving structural integrity through hydrocolloidal gel formation and moisture entrapment; and

(2) Nutritional: by introducing natural antioxidants and omega-3 fatty acids that mitigate oxidation and improve overall product stability.

These outcomes validate chia seed extract as a promising clean-label alternative to synthetic antioxidants (e.g., BHT, BHA) in processed meats. Beyond yield enhancement, the presence of chia bioactives contributes to extended shelf-life, improved sensory attributes, and a more sustainable, health-conscious product formulation [44, 45]. Future studies exploring the molecular interactions between chia polysaccharides and meat proteins, as well as the sensory perception of CSE-enriched products, will be essential to further optimize formulation levels and validate consumer acceptance. Nonetheless, the present findings clearly establish that chia seed extracts, particularly at higher inclusion levels (T3), offer substantial benefits in terms of cooking performance, product yield, and storage stability—confirming their suitability as multifunctional ingredients in modern meat processing.

3.2.4 Texture Attributes

Texture is one of the most critical determinants of meat product quality, influencing both consumer acceptability and industrial performance. In this study, the influence of chia seed extract (CSE) on the texture profile of beef burgers was evaluated through five key parameters—hardness, springiness, cohesiveness, chewiness, and resilience—over 15 days of refrigerated storage at 4 °C. A three-dimensional (3D) response surface methodology (RSM) approach was employed to visualize the interactive effects of CSE concentration and storage duration on texture characteristics (Fig. 3A–E). This analytical approach provided an integrated understanding of how chia extracts, through their hydrocolloid-forming capacity and bioactive compounds, modulate the mechanical behavior of meat matrices during storage (Table 8). Table 8 presents the evolution of texture parameters in beef burgers supplemented with different concentrations of CSE (T1–T3), compared to the control (without additives) and SET (synthetic antioxidant-treated) samples. Texture attributes are vital indicators of the structural integrity, protein–water interactions, and fat-binding capacity of the meat system. These properties can deteriorate during cold storage due to protein denaturation, lipid oxidation, and moisture migration. However, the addition of CSE, rich in phenolics, polysaccharides, and mucilage, may mitigate these changes and improve texture stability through its antioxidant and water-retention capabilities.

Fig. 3.

Three-dimensional (3D) response surface plots from texture profile analysis of beef burgers prepared with chia seed extracts during 15 days of cold storage at 4 °C. (A) Hardness: Illustrates the interactive effects of CSE concentration and storage duration on burger firmness. (B) Springiness: Shows how CSE concentration and storage time influence the elastic recovery of burgers. (C) Cohesiveness: Depicts the combined effects of CSE and storage on internal structural integrity. (D) Chewiness: Represents the influence of CSE and storage duration on the energy required to chew the burgers. (E) Resilience: Displays the effect of CSE and storage on the ability of burgers to recover their shape after deformation.

Table 8. Texture profile analysis (TPA) of beef burgers prepared with chia seed extracts during cold storage at 4 °C for 15 days.

Parameter	Day	Control	SET	T1	T2	T3
Hardness (N)	0	12.41 $\pm$ 0.71 abA	11.73 $\pm$ 1.02 abA	9.35 $\pm$ 0.54 cA	12.09 $\pm$ 0.85 abA	12.43 $\pm$ 0.91 aA
	5	15.62 $\pm$ 0.99 aA	12.91 $\pm$ 0.83 bA	9.26 $\pm$ 0.66 cA	14.93 $\pm$ 1.00 aA	12.86 $\pm$ 1.32 bA
	10	14.90 $\pm$ 0.91 aA	13.21 $\pm$ 0.76 abA	10.01 $\pm$ 0.68 cA	14.43 $\pm$ 1.31 abA	13.86 $\pm$ 0.88 abA
	15	13.43 $\pm$ 0.93 aA	12.99 $\pm$ 1.05 aA	10.35 $\pm$ 0.61 bA	13.42 $\pm$ 0.96 aA	11.49 $\pm$ 0.79 bA
Springiness (mm)	0	6.30 $\pm$ 0.11 aA	6.42 $\pm$ 0.17 aA	6.40 $\pm$ 0.09 aA	6.48 $\pm$ 0.14 aA	6.35 $\pm$ 0.16 aA
	5	6.36 $\pm$ 0.15 aA	6.40 $\pm$ 0.13 aA	6.32 $\pm$ 0.12 aA	6.33 $\pm$ 0.17 aA	6.38 $\pm$ 0.08 aA
	10	6.34 $\pm$ 0.18 aA	6.43 $\pm$ 0.15 aA	6.36 $\pm$ 0.11 aA	6.10 $\pm$ 0.19 aA	6.37 $\pm$ 0.10 aA
	15	6.29 $\pm$ 0.13 aA	6.28 $\pm$ 0.10 aA	6.41 $\pm$ 0.13 aA	6.40 $\pm$ 0.12 aA	6.39 $\pm$ 0.09 aA
Cohesiveness	0	0.87 $\pm$ 0.05 bB	1.00 $\pm$ 0.04 aA	0.99 $\pm$ 0.02 aA	0.93 $\pm$ 0.03 abA	0.96 $\pm$ 0.02 abA
	5	0.91 $\pm$ 0.03 bAB	0.99 $\pm$ 0.03 aA	0.94 $\pm$ 0.03 abA	0.95 $\pm$ 0.04 abA	0.95 $\pm$ 0.04 abA
	10	0.94 $\pm$ 0.02 aA	0.96 $\pm$ 0.02 aA	0.95 $\pm$ 0.03 aA	0.96 $\pm$ 0.04 aA	0.94 $\pm$ 0.05 aA
	15	0.96 $\pm$ 0.03 aA	0.99 $\pm$ 0.03 aA	0.94 $\pm$ 0.03 abA	0.97 $\pm$ 0.02 aA	0.94 $\pm$ 0.04 abA
Chewiness (N·mm)	0	68.24 $\pm$ 3.02 aA	75.12 $\pm$ 3.22 aA	61.18 $\pm$ 2.45 aA	72.46 $\pm$ 2.97 aA	73.94 $\pm$ 3.10 aA
	5	70.12 $\pm$ 2.88 aA	73.33 $\pm$ 3.12 aA	62.43 $\pm$ 2.58 aA	75.58 $\pm$ 2.74 aA	72.11 $\pm$ 2.61 aA
	10	72.40 $\pm$ 3.17 aA	71.36 $\pm$ 2.89 aA	65.29 $\pm$ 2.91 aA	74.11 $\pm$ 2.65 aA	70.34 $\pm$ 2.93 aA
	15	71.31 $\pm$ 2.96 aA	72.05 $\pm$ 3.01 aA	66.52 $\pm$ 2.73 aA	71.87 $\pm$ 3.10 aA	68.74 $\pm$ 2.88 aA
Resilience	0	0.44 $\pm$ 0.02 abA	0.46 $\pm$ 0.01 aA	0.43 $\pm$ 0.01 abA	0.48 $\pm$ 0.02 aA	0.42 $\pm$ 0.01 bA
	5	0.45 $\pm$ 0.02 aA	0.47 $\pm$ 0.01 aA	0.44 $\pm$ 0.01 abA	0.45 $\pm$ 0.02 aA	0.41 $\pm$ 0.02 bA
	10	0.43 $\pm$ 0.02 bA	0.45 $\pm$ 0.01 abA	0.42 $\pm$ 0.01 bA	0.44 $\pm$ 0.02 abA	0.38 $\pm$ 0.01 cA
	15	0.44 $\pm$ 0.01 abA	0.46 $\pm$ 0.01 aA	0.43 $\pm$ 0.01 abA	0.45 $\pm$ 0.01 aA	0.41 $\pm$ 0.01 bA

Notes:

$\bullet$ Values are expressed as mean $\pm$ SD (n = 3).

$\bullet$ Different lowercase letters (a–c) in the same row indicate significant differences among treatments at a specific storage day (p $\le$ 0.05, Tukey’s test).

$\bullet$ Different uppercase letters (A–B) in the same column indicate significant differences across storage periods within the same treatment (p $\le$ 0.05, Tukey’s test).

$\bullet$ T1–T3 represent different concentrations of chia seed extract. SET, synthetic antioxidant-treated group (positive control).

Hardness values varied significantly (p $\le$ 0.05) between treatments and across storage periods. At day 0, the control and SET groups exhibited higher hardness (12.41 $\pm$ 0.71 N and 11.73 $\pm$ 1.02 N, respectively), while T1 (9.35 $\pm$ 0.54 N) was markedly softer. Treatments T2 and T3 maintained hardness levels (12.09 $\pm$ 0.85 N and 12.43 $\pm$ 0.91 N) comparable to the control, suggesting that moderate-to-high concentrations of chia extract support an appropriate structural firmness. Over time, hardness generally increased, reflecting the natural tendency of meat proteins to crosslink and lose moisture during refrigeration. However, the extent of hardening was attenuated in CSE treatments, particularly in T3 (11.49 $\pm$ 0.79 N) after 15 days, compared to the control (13.43 $\pm$ 0.93 N). This indicates that chia’s hydrocolloid fraction and phenolic antioxidants preserve juiciness and delay protein aggregation, maintaining desirable texture stabilit-y (Table 8).

Fig. 3A, the three-dimensional response surface plot for hardness illustrates the interactive effects of chia seed extract (CSE) concentration and storage duration on the firmness of beef burgers during 15 days of refrigerated storage at 4 °C. A gradual decline in hardness was observed in all formulations over time, reflecting typical softening associated with protein denaturation, moisture migration, and weakening of the myofibrillar network during cold storage [35, 42]. Notably, burgers fortified with moderate CSE levels (T2: 1.0%) maintained hardness values comparable to the control, indicating good structural preservation. In contrast, higher inclusion (T3: 1.5%) produced a slightly softer texture (p 0.05) at extended storage times, likely due to the water-binding and gel-forming properties of chia mucilage, which impart greater moisture retention and elasticity to the meat matrix. These results demonstrate that CSE concentration modulates texture in a controllable manner, allowing formulation flexibility for different product profiles.

This firmness retention effect is mainly linked to the hydrocolloid-forming capacity of chia mucilage and its high dietary fiber content, which together generate a gel-like network within the meat matrix. This structure improves water-holding capacity (WHC) and minimizes structural disintegration during storage [35, 42, 48]. Furthermore, chia-derived phenolics—particularly gallic, ferulic, and caffeic acids—are likely to form non-covalent and covalent interactions with muscle proteins and connective tissue, contributing to cross-linking that reinforces matrix integrity and resists oxidative or enzymatic degradation [35, 42].

Conversely, hardness in the control and SET samples decreased more rapidly, reflecting the absence of hydrocolloid reinforcement and limited oxidative protection. These findings agree with previous reports indicating that plant extracts rich in polyphenols and soluble fibers synergistically preserve textural stability and extend the sensory shelf-life of meat products during refrigerated storage [49]. Collectively, the enhanced hardness retention in CSE-enriched burgers underscores the multifunctional role of chia extract as both an antioxidant and a natural hydrocolloid, thereby improving product quality and consumer acceptability throughout cold storage.

Springiness values were relatively consistent across treatments and days (6.10–6.48 mm), showing no significant differences (p $>$ 0.05). This stability implies that chia extract incorporation did not disrupt the product’s ability to regain its shape after compression. The presence of soluble fiber and mucilage in chia may have reinforced the elastic gel network, preserving the structural recovery of the protein matrix even under cold storage. Such findings agree with previous reports indicating that hydrocolloids from plant sources enhance elasticity and water retention in emulsified meat systems (Table 8).

The response surface plot for springiness (Fig. 3B) shows that no significant differences (p $>$ 0.05) were observed among treatments throughout the 15 days of refrigerated storage at 4 °C. This overall stability indicates that the elastic properties of beef burgers were effectively maintained during storage, despite the minor, non-significant downward trends typically associated with protein denaturation, moisture redistribution, and reduced muscle fiber elasticity during cold storage [35, 42, 50]. Although the differences were not statistically significant, burgers containing chia seed extract (CSE) at 1.0 % (T2) and 1.5 % (T3) tended to show slightly higher springiness values than the control and synthetic antioxidant-treated (SET) samples. This observation suggests that CSE may exert a stabilizing effect on the meat matrix, likely due to its mucilage and polyphenol content, which enhance water retention and matrix elasticity. Overall, the inclusion of CSE contributed to maintaining textural resilience and uniformity during refrigerated storage.

This effect can be attributed to the hydrophilic polysaccharides in chia mucilage, which form a gel-like network within the meat matrix, entrapping water and reinforcing protein interactions, thereby minimizing structural collapse [44, 45, 51]. Additionally, the presence of polyphenolic and flavonoid compounds such as quercetin, rutin, and chlorogenic acid may inhibit protein oxidation, preserving the integrity of myofibrillar proteins responsible for elasticity [35, 42]. The stable springiness observed during storage highlights chia seed extract’s multifunctional ability to sustain mechanical strength and enhance the perceived juiciness and freshness of beef burgers during cold storage.

Cohesiveness describes the internal bonding strength of the product’s matrix. At day 0, the control exhibited the lowest cohesiveness (0.87 $\pm$ 0.05), whereas SET and chia-treated samples (T1–T3) displayed higher values (0.93–1.00), demonstrating improved internal integrity due to chia components. Cohesiveness remained stable (0.94–0.99) throughout the 15-day period, suggesting that chia extracts provided a protective effect against oxidative and enzymatic degradation of structural proteins. This may be attributed to the phenolic–protein interactions and the formation of hydrated networks by chia mucilage, both of which enhance binding within the muscle fiber matrix (Table 8).

The response surface plot for cohesiveness (Fig. 3C) demonstrates that the changes in all treatments during 15 days of refrigerated storage were not statistically significant (p $>$ 0.05). This suggests that the internal binding strength and structural integrity of the burgers remained relatively stable throughout storage. Nonetheless, burgers fortified with chia seed extract (CSE) at 1.0 % and 1.5 % (T2 and T3) consistently showed higher cohesiveness values than the untreated control and synthetic antioxidant-treated (SET) samples, indicating that CSE contributed to maintaining the internal structure of the meat matrix.

Cohesiveness in meat systems primarily depends on protein–protein and protein–water interactions, which can deteriorate under oxidative or enzymatic stress [45, 52]. The enhanced cohesiveness observed in CSE-fortified burgers can be attributed to the dual functionality of chia seed components: (i) phenolic acids such as gallic, ferulic, and caffeic acids protect against protein oxidation by minimizing carbonyl formation and preserving sulfhydryl groups [39, 46], and (ii) chia mucilage, rich in soluble polysaccharides, forms hydrocolloid-like gels that fill interstitial spaces, enhance water binding, and stabilize the protein network [47]. Overall, the maintenance of cohesiveness during storage underscores chia seed extract’s structural and antioxidative roles, improving the texture, juiciness, and handling properties of beef burgers without significant degradation over time.

Chewiness, a function of hardness, cohesiveness, and springiness, reflects the energy required to masticate the sample. Initially, chewiness values ranged from 61.18 N$\cdot$mm (T1) to 75.12 N$\cdot$mm (SET), with minimal variation (p $>$ 0.05) among groups. Across storage, chewiness remained relatively stable for all treatments, indicating that chia supplementation did not negatively affect mouthfeel or chew resistance. After 15 days, chewiness values were 71.31 N$\cdot$mm (control), 72.05 N$\cdot$mm (SET), and 66.52–71.87 N$\cdot$mm for chia-treated samples. These results show that CSE inclusion maintained mechanical integrity while possibly improving juiciness perception. The hydrocolloid matrix formed by chia’s polysaccharides likely contributed to a smoother, less brittle texture by retaining water within the protein network, counteracting the toughening effects of oxidation and dehydration typically observed in untreated burgers (Table 8 and Fig. 3D).

Resilience measures the product’s ability to recover energy after deformation, thus reflecting structural elasticity. Values ranged between 0.38 and 0.48 across treatments. T2 displayed the highest resilience (0.48 $\pm$ 0.02 at day 0), indicating a more elastic and compact network. Conversely, slightly lower values were recorded for T3 (0.42 $\pm$ 0.01), suggesting that higher chia levels may increase moisture retention but reduce elastic energy recovery due to a softer matrix. Despite minor fluctuations, resilience remained comparable to or higher than the control during storage, confirming chia’s ability to stabilize the structural framework of the meat product (Table 8). The response surface plot for resilience (Fig. 3E) and the corresponding data in Table 8 demonstrated no significant variation (p $>$ 0.05) across storage time, indicating that the ability of the meat matrix to recover its shape after deformation remained largely stable throughout the 15 days of refrigerated storage. Resilience reflects the elastic recovery and internal cohesion of the protein gel network after compression, parameters that depend on protein–protein interactions, water-binding capacity, and the integrity of the myofibrillar matrix.

Across all treatments, resilience values ranged narrowly between 0.38 and 0.48, with no statistically significant changes over time (same uppercase letter “A” in each column). Burgers supplemented with 1.0% CSE (T2) consistently exhibited slightly higher resilience values compared to the control and other treatments, suggesting that moderate levels of chia seed extract enhanced the elastic recovery of the meat structure. The 1.5% CSE (T3) group showed comparable resilience stability, indicating that higher chia inclusion did not compromise textural integrity.

This relative stability can be attributed to the hydrocolloid-forming polysaccharides present in chia mucilage, which interact with the meat matrix to strengthen the gel network and help retain water. Additionally, the antioxidant phenolic compounds identified in the extract—particularly gallic, ferulic, caffeic, and quercetin—may have contributed to maintaining protein functionality by limiting oxidative damage to myofibrillar proteins [49, 50, 51]. The combined hydrocolloid and antioxidant mechanisms likely prevented excessive protein cross-linking or degradation, thereby preserving textural elasticity during cold storage.

From a technological standpoint, the stability of resilience across all formulations indicates that chia seed extract effectively supports the mechanical structure of beef burgers without inducing textural deterioration. This is particularly relevant for refrigerated meat systems, where protein oxidation and moisture loss typically reduce elasticity over time. Therefore, the inclusion of CSE not only maintains the natural rebound capacity of the meat matrix but also contributes to the overall textural and structural quality of the product.

Collectively, these findings confirm that chia seed extract functions as a natural stabilizer, preserving resilience and ensuring that the final product retains desirable firmness and elasticity throughout storage. Such stability reinforces its potential as a clean-label antioxidant and texture-enhancing agent in reformulated meat products aimed at extending shelf-life while maintaining consumer-acceptable texture profiles.

Overall Evaluation: The incorporation of chia seed extracts effectively enhanced or preserved the texture of beef burgers during refrigerated storage. Treatments containing moderate concentrations (T2) achieved the most favorable balance between firmness, elasticity, and cohesiveness, whereas high concentrations (T3) resulted in slightly softer but juicier products. Chia’s physicochemical properties—especially its soluble fiber and mucilage—contribute to improved water-holding capacity and gel-forming potential, preventing excessive hardening and moisture loss over time. Furthermore, chia’s abundant phenolic antioxidants likely inhibited protein oxidation and crosslinking, thus maintaining a stable texture profile during storage. From a technological standpoint, these findings demonstrate that chia seed extract functions as a natural texture stabilizer comparable to synthetic antioxidants, with added benefits of water retention and oxidative protection. This aligns with the growing demand for clean-label, plant-derived additives in processed meat formulations. The ability of chia components to maintain consistent hardness, chewiness, and cohesiveness over 15 days indicates their synergistic action between hydrocolloid and antioxidant properties. Overall, the inclusion of chia seed extracts, particularly at intermediate levels (T2), provided significant texture preservation without compromising sensory attributes. This suggests potential application in reformulated meat systems aimed at improving shelf-life, juiciness, and structural stability using natural alternatives to synthetic stabilizers. Future work should focus on correlating these textural changes with microstructural and rheological analyses to elucidate the mechanisms of chia–protein interactions responsible for the observed improvements. These 3D RSM plots clearly demonstrate that chia seed extract (CSE) not only enhances oxidative stability but also plays a pivotal role in preserving the textural integrity of beef burgers during refrigerated storage. The synergistic functionality of chia mucilage, dietary fiber, and polyphenolic compounds underscores its value as a multifunctional, clean-label additive for meat processing. Among the tested inclusion levels, 1.0% CSE (T2) provided the most balanced texture profile, maintaining the highest resilience and overall textural stability during storage, whereas 1.5% CSE (T3) exhibited slightly lower resilience values but retained desirable firmness and structural integrity. The T1 formulation produced softer textures, suitable for applications requiring enhanced tenderness, while T3 imparted greater hardness and cohesiveness. Cohesiveness and springiness remained largely stable (p $>$ 0.05) across storage for all formulations, confirming that chia extracts sustain product integrity without undesirable textural changes. Collectively, these findings emphasize the dual functionality of chia extracts as natural antioxidants and texturizing agents, contributing to improved sensory and technological quality during chilled storage. The application of response surface methodology (RSM) provided an effective visualization of these interactions, supporting optimized formulation strategies for functional, clean-label meat products.

Lipid oxidation is one of the most critical factors affecting the quality, safety, and shelf-life of meat products during storage. It leads to the development of rancid flavors, discoloration, and nutritional deterioration. The thiobarbituric acid reactive substances (TBARS) assay was used to assess the extent of lipid oxidation in beef burgers formulated with chia seed extract (CSE) at varying concentrations (T1–T3) and stored at 4 °C for 15 days. The corresponding data are presented in Table 9. At the beginning of storage (day 0), the malonaldehyde (MDA) content in the control burgers was 0.47 mg/kg, which increased sharply to 2.52 mg/kg by day 10 before slightly declining to 1.27 mg/kg at day 15. This pattern reflects the typical progression of lipid oxidation, where primary oxidation products (hydroperoxides) accumulate and subsequently decompose into secondary aldehydes such as MDA. The increase observed in the control samples was statistically significant (p $\le$ 0.05), confirming that the absence of antioxidants allowed extensive oxidative degradation during storage.

In contrast, burgers containing chia seed extract (CSE) or synthetic antioxidant (SET) displayed markedly lower TBARS values across all time points, indicating effective oxidative stabilization. The SET-treated samples showed only minimal changes (0.27–0.38 mg/kg) over 15 days, consistent with its known antioxidant potency. Among the CSE formulations, the T3 treatment (high CSE) exhibited the lowest MDA concentrations throughout storage, ranging from 0.21 to 0.36 mg/kg.

The T2 formulation (medium CSE) also effectively delayed lipid peroxidation, showing only minor fluctuations from 0.23 mg/kg at day 0 to 0.59 mg/kg at day 15. Although there was a slight upward trend, the changes over time were not statistically significant until the final sampling point (A–B superscripts), suggesting that moderate chia extract concentrations are sufficient to retard oxidation over two weeks of refrigerated storage.

Importantly, the T1 formulation (low CSE) maintained statistically similar MDA values throughout storage (all labeled “A”), indicating no significant increase (p $>$ 0.05) from day 0 to day 15. This stability implies that even at lower doses, chia seed extract provides substantial antioxidative protection, likely due to its rich phenolic composition and synergistic interaction between polyphenols, fiber, and mucilage components. The bioactive compounds identified in Tables 3,4—such as chlorogenic acid, caffeic acid, ferulic acid, rutin, and quercetin—are known for their radical-scavenging and metal-chelating activities, which collectively suppress peroxidation in lipid-rich matrices.

Among all treatments, the control group exhibited the highest degree of oxidation, while T3 (high CSE) and SET (synthetic antioxidant) performed best in preserving oxidative stability. The low TBARS values in T3 samples across all time points ($\le$0.36 mg/kg) suggest that chia extract at higher concentrations can effectively maintain lipid integrity during cold storage. These findings align with previous reports demonstrating that chia-derived polyphenols and mucilage compounds act as natural antioxidants by donating hydrogen atoms to neutralize free radicals, stabilizing peroxides, and forming protective barriers that limit oxygen diffusion into the meat matrix.

Mechanistically, the antioxidant activity of CSE can be attributed to both its hydrophilic and lipophilic constituents. The hydrophilic phenolics quench reactive oxygen species (ROS) in the aqueous phase, while the viscous chia mucilage forms a network that restricts lipid mobility and oxygen penetration. This dual mode of action results in a consistent reduction of oxidation products and enhances the overall oxidative stability of the burgers. Additionally, chia seed’s high content of $\alpha$-linolenic acid, while prone to oxidation, may interact with its endogenous antioxidants to create a balanced redox environment, further enhancing lipid protection.

Overall, the results from Table 9 confirm that chia seed extract significantly inhibits lipid oxidation in beef burgers during refrigerated storage. The efficacy increases with extract concentration, with T3 providing the most robust antioxidative effect. The absence of significant differences over time in the T1 and T2 groups underscores the stability and sustained antioxidant potential of chia phenolics even at moderate dosages. From a formulation perspective, CSE offers a promising natural alternative to synthetic antioxidants, aligning with clean-label and functional food trends. The use of chia seed extract not only supports oxidative stability but also complements previous findings (Tables 6,7,8) showing improvements in cooking yield and textural quality.

Thus, chia seed extract can be considered a multifunctional ingredient—providing both antioxidant protection and technological benefits—making it highly suitable for the development of healthier, more stable meat products.

The observed antioxidant effect can be attributed to chia’s rich bioactive composition, including polyphenols (such as gallic, caffeic, and ferulic acids), flavonoids (notably quercetin and rutin), and $\alpha$-linolenic acid, all of which exert radical-scavenging, metal-chelating, and lipid-protective actions [53, 54, 55]. These compounds synergistically stabilize the lipid matrix by neutralizing reactive oxygen species, chelating transition metals that catalyze oxidation, and enhancing membrane integrity through amphiphilic interactions. Notably, while the synthetic antioxidant-treated group (SET) also effectively suppressed lipid peroxidation, its performance was occasionally surpassed by T3, particularly at the later stages of storage. This observation underscores the potent and sustained antioxidant activity of chia seed extract, likely due to its combination of phenolic acids and mucilage compounds that provide both hydrophilic and lipophilic protection. Consequently, CSE not only matches but in some cases exceeds the efficacy of conventional synthetic antioxidants in maintaining lipid stability in meat systems. These results reinforce the potential of chia seed extract as a natural, multifunctional preservative capable of replacing synthetic additives while enhancing both the nutritional and functional quality of processed meats.

These findings are consistent with recent evidence showing that plant-derived antioxidants, when applied at effective concentrations, can rival or even surpass the activity of synthetic preservatives such as BHT and BHA [54, 55]. Moderate CSE inclusion levels (T1 and T2) provided intermediate protection, with TBARS values remaining below the rancidity threshold but increasing more gradually over storage. This outcome reinforces the dose-dependent antioxidant capacity of chia extracts. The steady rise in TBARS across all treatments—most pronounced in the control and lower CSE levels—reflects the inherent progression of oxidative reactions in refrigerated meat systems, primarily driven by the abundance of unsaturated fatty acids, oxygen exposure, and iron-catalyzed prooxidant activity [56].

Beyond direct oxidative control, CSE may also enhance water retention and structural stability by forming a hydrophilic fiber–mucilage network that limits oxygen diffusion and delays lipid degradation. This dual protective mechanism highlights CSE’s multifunctionality, simultaneously improving oxidative resistance and maintaining textural quality during storage. From a technological and commercial standpoint, these attributes are highly relevant: the incorporation of CSE not only prolongs the shelf-life of meat products but also aligns with the growing demand for clean-label, naturally preserved foods. Its dual role as a preservative and functional ingredient, coupled with its rich nutraceutical profile, positions chia extract as a promising additive for developing healthier meat formulations targeted at quality-driven and health-conscious consumer markets [56, 57, 58].

While TBARS analysis provided clear evidence of lipid stabilization by chia seed extract, it primarily reflects secondary oxidation products. Future studies should incorporate complementary endpoints such as protein carbonyl quantification, volatile aldehydes (e.g., hexanal by GC–MS), and metal chelation assays to capture a more comprehensive picture of oxidative processes and confirm the dual lipid–protein protective effects of the extract.

In addition to the observed antioxidative and physicochemical benefits, future research should also include systematic microbiological assessments (total viable counts, psychrotrophs, Enterobacteriaceae, and yeasts/molds) under different storage and packaging systems (aerobic, vacuum, and MAP). This would provide a more comprehensive evaluation of chia seed extract’s contribution to both chemical stability and microbiological safety in extending the shelf-life of beef burgers.

It should be noted that polyphenols in Salvia hispanica extract may chelate transition metals, potentially influencing non-heme iron bioaccessibility. Although the concentrations applied in this study align with clean-label practices, future research should evaluate iron bioavailability and heme stability to ensure that antioxidant benefits are not accompanied by unintended nutritional trade-offs.

Within the tested inclusion levels, chia seed extract (CSE) proved technologically effective in enhancing oxidative stability, color retention, and textural attributes of beef burgers while maintaining sensory acceptability. These results support its compatibility with clean-label objectives, providing a natural alternative to synthetic antioxidants such as BHT and BHA. Nevertheless, broader considerations are needed to consolidate its application in commercial systems. Standardization of the extract, including marker identification and batch-to-batch reproducibility, will be essential for industrial reliability. Additionally, further studies should define the optimal balance between antioxidant efficacy and sensory thresholds, supported by consumer acceptance testing. Expanding oxidative stability assessments to include protein oxidation and volatile compounds, alongside systematic microbiological monitoring under different packaging conditions, will provide a more complete picture of shelf-life extension. Safety aspects, including potential allergenicity, antinutritional factors, and effects on iron bioavailability, must also be evaluated to ensure nutritional adequacy. Finally, pilot-scale studies across different meat matrices (e.g., poultry, pork) and fat levels will help validate scalability and generalisability. Overall, the present findings highlight CSE as a promising candidate for clean-label meat preservation, with future work focused on regulatory compliance and industrial feasibility to support broader adoption.