Mediterranean Mussel (Mytilus galloprovincialis) Byssus Ameliorates Diet-Induced Metabolic Syndrome in High-Fat High-Sucrose-Fed Rats by Reinforcing Antioxidant Defenses and Attenuating Systemic Inflammation

Abstract

Byproducts of marine invertebrate-based industries represent an underexplored reservoir of bioactive compounds with therapeutic potential. This study investigated the efficacy of Mediterranean mussel (Mytilus galloprovincialis) byssus against diet-induced metabolic syndrome, with emphasis on the mechanisms of its antioxidant and anti-inflammatory activities. To establish metabolic dysregulation, 24 male Wistar rats were fed a high-fat, high-sucrose diet for 12 weeks, followed by a 28-day intervention with 12% byssus supplementation. Byssus significantly corrected the dyslipidemic profile, reducing low-density lipoprotein cholesterol, triglycerides, and total cholesterol while elevating the cardioprotective high-density lipoprotein cholesterol (P<0.001). Glycemic control improved significantly, with reductions in fasting and postprandial glucose (P<0.05), accompanied by a favorable downward trend in glycated hemoglobin, while circulating insulin levels remained stable throughout the intervention. Hepatocellular injury was alleviated, as reflected by significant decreases in serum alanine aminotransferase and aspartate aminotransferase activities (P<0.001). These benefits were accompanied by a broad reinforcement of endogenous antioxidant defenses, including a notable upregulation of superoxide dismutase across serum, erythrocytes, and liver, with a significant increase observed at the hepatic level (P<0.05), and robust restoration of hepatic catalase (P<0.01) and glutathione reductase (P<0.001) activities compared to the untreated HFHS group, alongside a substantial recovery of paraoxonase-1 arylesterase activity that approached, though did not fully reach, control levels (P<0.001). Concurrently, tumor necrosis factor-α was significantly reduced (P<0.01), alongside a favorable decreasing trend in interleukin-6 and C-reactive protein, reflecting the suppression of chronic inflammation associated with metabolic syndrome. These findings suggest that M. galloprovincialis byssus exerts marked hypolipidemic, hypoglycemic, and hepatoprotective influence, potentially associated with the reinforcement of redox homeostasis and attenuation of systemic inflammation, supporting its potential as a marine-derived functional ingredient for the adjunctive management of metabolic syndrome.

INTRODUCTION

As a vital pillar of metabolic health, carbohydrate homeostasis relies on the finely tuned orchestration of liver-peripheral tissue glucose fluxes, which, when compromised, cause hyperglycemia that transitions into a state of chronic glucotoxicity. The global prevalence of this metabolic disorder is increasing at an alarming, epidemic rate. This phenomenon functions as a relentless molecular stressor, shifting from a simple glycemic imbalance to a systemic mediator of cell damage and metabolic instability (Banday et al., 2020). It is associated with deleterious structural and functional alterations driven by non-enzymatic protein glycation and the accumulation of advanced glycation end products, ultimately irreversibly damaging tissues (Ferrannini, 2021). Within this clinical framework, postprandial hyperglycemia plays a pivotal role. Acute and oscillatory glycemic patterns induce more severe endothelial dysfunction than sustained basal hyperglycemia, primarily by abruptly overwhelming endogenous antioxidant defense systems and exacerbating oxidative stress (Maruhashi and Higashi, 2021).

Pathophysiologically, glucotoxicity rarely develops in isolation but is closely intertwined with lipotoxicity, typically characterized by dyslipidemic hyperlipidemia, including elevated triglycerides (TGs) and low density lipoprotein-cholesterol (LDL-C) levels (Petrie et al., 2018). Excess lipid intake can trigger hepatic accumulation and oxidative responses, leading to metabolic disturbances and contributing to hepatic steatosis and obesity, thereby increasing the risk of chronic metabolic complications (Daidj and Lamri-Senhadji, 2021). Such coexistence impaired metabolic flexibility, simultaneously overloading carbohydrate and lipid substrates, saturating the mitochondrial respiratory chain. As a consequence, excessive electron leakage occurs, amplifying the production of reactive oxygen species (ROS) and positioning mitochondria as epicenters of cellular oxidative stress (Yan, 2014).

This process is sustained by a bidirectional amplification loop involving oxidative stress and low-grade chronic inflammation. ROS generated under hyperglycemia and hyperlipidemia activate pro-inflammatory transcription factors, notably nuclear factor-κB (NF-κB) (Maruhashi and Higashi, 2021). These further promote the release of inflammatory cytokines, which disrupt insulin signaling pathways and perpetuate metabolic dysfunction, forming a self-reinforcing vicious cycle involving pathological progression (Yan, 2014).

Current pharmacological strategies partially control glycemic and lipid parameters, but their long-term efficacy is frequently limited by clinically relevant iatrogenic impacts, including myopathic and hepatic complications as well as gastrointestinal intolerance, which collectively compromise patient adherence (Banday et al., 2020). These limitations have prompted growing interest in developing alternative therapeutic approaches based on natural bioactives. In this context, marine resources have emerged as particularly promising candidates because of their capacity to exert a pleiotropic influence on glucose and lipid metabolism while offering a favorable safety profile (Xiang et al., 2022).

Bivalves have an annual global production exceeding 15 million tons; their associated industries generate large quantities of byproducts, such as byssus and viscera, rich in proteins, peptides, and polysaccharides (Neves et al., 2016; Wijsman et al., 2019). Among these, the byssus of Mediterranean mussels (Mytilus galloprovincialis) is particularly notable for its elevated contents of multidomain pre-collagenous proteins－PreCol-D, PreCol-NG, and PreCol-P－and type I collagen organized into a characteristic Gly-Pro-X triple-helical structure, where X may correspond to any amino acid, most frequently alanine, lysine, arginine, or aspartic acid, as well as post-translationally modified residues like 3,4-dihydroxyphenylalanine (DOPA). Such a unique molecular organization confers exceptional mechanical stability and resilience in marine environments (Lassoued et al., 2016; Jehle et al., 2021; Renner-Rao et al., 2024). Unlike purified collagen hydrolysates, the raw byssal exoskeleton preserves the intactness of triple-helical domains as well as non-collagenous motifs, suggesting a biopotency comparable to that of native marine collagen. The bioeffects of byssus may therefore be attributed, at least in part, to the generation of collagen-derived bioactive peptides, as reported for other marine sources (Golser and Scheibel, 2017; Silva et al., 2021). Although the byssus of M. galloprovincialis has been extensively studied for its remarkable mechanical properties, the potential therapeutic relevance in metabolic disorders remains poorly explored, especially for dyslipidemia and glycemic regulation, including postprandial responses.

This study assessed the in vivo impacts of supplementation with M. galloprovincialis byssus on glucose and lipid homeostasis in Wistar rats fed a high-fat, high-sucrose (HFHS) diet. Antioxidant and anti-inflammatory mechanisms, which may underlie improvements in diet-induced metabolic disturbances, received special attention.

MATERIALS AND METHODS

Collection of Mediterranean mussels, preparation, and recovery of byssus

Adult Mediterranean mussels were manually collected at depths of 0－10 m during December 2025 from rocky substrates in the Kristel port area, Oran Province, Algeria (35.822932° N; −0.490610° W). The specimens selected exhibited an average shell length of 32.50±4.75 and a mean total body weight of 11.2±1.5 g. The mussels were transported on ice to the Laboratory of Clinical and Metabolic Nutrition, University of Oran 1 Ahmed Ben Bella, Algeria, and washed with distilled water to remove debris. The soft tissues and shells were then separated, and the byssus threads were carefully isolated from the foot. The byssus was cleaned, air-dried at room temperature (∼25°C) for 72 h until a constant weight was reached, ground into a fine powder, sieved, and finally stored in a desiccator under dry conditions.

Nutritional composition of Mediterranean mussels byssus

The chemical composition of byssus was determined using standard AOAC methods (Horwitz, 2005). Proteins were quantified employing the Kjeldahl method using a nitrogen-to-protein conversion factor of 6.25, in accordance with the AOAC Official Method 984.13 (Kjeldahl, 1883). Notably, this factor may slightly overestimate the protein content due to the presence of non-proteinaceous nitrogen-containing compounds, including catecholic residues such as DOPA, characteristic of byssal adhesive proteins. Total lipids were extracted per the Folch method (Folch et al., 1957) employing a chloroform-methanol (2:1, v/v) mixture. Ash content was determined via incineration at 550°C for 4 h; complete incineration was confirmed by the absence of black particles in the ash (Horwitz, 2005). Moisture was measured by oven-drying at 105°C until constant weight (Horwitz, 2005). Carbohydrate content was calculated by the difference in carbohydrate (%)=100%−[protein (%)+total lipids (%)+ash (%)+water (%)] (Horwitz, 2005) and ascertained spectrophotometrically by applying the phenol-sulfuric acid method (DuBois et al., 1956), with glucose as an external standard.

Collagen extraction from byssus

Pepsin-solubilized collagen (PSC) was extracted from raw byssus powder according to the protocol described by Rodríguez et al. (2017), optimized by evaluating a fixed enzyme-to-substrate (E/S) ratio over two hydrolysis periods: 4 and 24 h. For each experimental trial, 12 g of powder was initially rehydrated in 240 mL of distilled water at 1:20 (w/v) under continuous stirring at 200 rpm and 4°C for 4 h. Non-collagenous proteins were removed through alkaline pre-treatment in 120 mL of a 0.1 N NaOH solution at 1:10 (w/v based on initial dry weight) for 1 h at 4°C. The matrix was demineralized by immersion in 120 mL of 0.1 N HCl (1:10, w/v based on initial dry weight) and agitated at 4°C for 18 h. After each pre-treatment, the pellet was recovered via centrifugation 5,000 g, 15 min, 4°C and repeatedly rinsed with deionized water until neutral pH.

To evaluate the effect of hydrolysis duration on collagen solubilization, the pre-treated pellets were resuspended in 120 mL of acidified water, adjusted to pH 1.5 with 5 N HCl, yielding a ratio of 1:10 (w/v relative to the initial dry weight) and enzymatically hydrolyzed using porcine gastric mucosa pepsin (Sigma-Aldrich) at a single E/S ratio of 1:20 (w/w, units/mg or weight ratio of pepsin to initial dry byssus weight). The extraction mixtures were constantly agitated at 25°C and evaluated at two incubation periods: 4 and 24 h.

Following incubation, the mixtures were centrifuged (10,000 g, 30 min, 4°C) to separate the solubilized collagen fraction and undigested residue. The solid residues were acid-extracted with 75 mL of 0.5 M acetic acid. The protein-enriched supernatants were combined, and the dissolved collagen was precipitated by adding NaCl to a final concentration of 0.7 M under slow stirring at 4°C for 12 h. The collagen precipitates obtained were isolated by high-speed centrifugation at 20,000 g for 1 h at 4°C, redissolved in 0.5 M acetic acid, and exhaustively purified via dialysis using 12－14 kDa molecular weight cut-off membranes (Sigma-Aldrich) against 0.5 M acetic acid and distilled water, for 48 h each, at 4°C. The final purified PSC isolates from the two durations were lyophilized and stored at −20°C. All optimization experiments were performed in triplicate (n=3).

Characterization of the PSC preparation

Quantification of total proteins: The total soluble protein content of the PSC was quantified using a colorimetric assay (Lowry et al., 1951), with minor modifications. Lyophilized PSC powder 10 mg was dissolved in 10 mL of ultrapure water to obtain a working solution at a concentration of 1 mg/mL. Bovine serum albumin (BSA) standards were prepared in parallel to construct an external calibration curve.

Briefly, the sample and standard aliquots were allowed to react with alkaline copper tartrate reagent for 10 min at room temperature, and were then mixed with Folin-Ciocalteu reagent. After incubation in the dark for 30 min, the A₇₅₀ was measured with a ultraviolet (UV)-Vis spectrophotometer against a reagent blank. Protein concentration was calculated by applying the calibration equation, y=0.3851x+0.0759 (R²=0.9826), where “y” and “x” represent the A₇₅₀ and protein concentration (mg/mL), respectively.

The total protein content of the PSC extract was calculated using the equation,

$$\text{Protein content (\%)}=\frac{\text{Protein concentration (mg/mL)}\times \text{Final sample volume (mL)}}{\text{Mass of initial PSC powder (mg)}}\times 100$$

All analyses were performed in triplicate (n=3).

Hydroxyproline (Hyp) and collagen content estimation

The Hyp content of the isolated PSC was determined utilizing a modified colorimetric method based on Woessner (1961) and adapted for microvolume analysis. Briefly, 10 mg of lyophilized PSC was hydrolyzed in 10 mL of 6 N HCl at 110°C for 24 h in vacuum-sealed borosilicate tubes. The samples were then cooled to room temperature; 100 µL of the hydrolysate was neutralized with 6 N NaOH and diluted to 10 mL with ultrapure water to a final pH of 7.0±0.2 and to ensure that the sample falls within the calibration curve’s linear range.

An external calibration curve was prepared using a high-purity L-Hyp standard over a concentration range of 0－2.5 µg/mL: 0, 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL. For the colorimetric assay, 100 µL of either the diluted sample or each standard solution was mixed with 100 µL of 14 mg/mL chloramine-T solution in 0.5 M acetate buffer (pH 6.0) and incubated at room temperature for 20 min to allow Hyp oxidation. Subsequently, 200 µL of 28 mg/mL Ehrlich’s reagent (p-dimethylaminobenzaldehyde) in isopropanol/perchloric acid (1:1, v/v) was added, and the reaction mixes were incubated in a thermostated water bath at 60°C for 20 min to develop the chromophore.

A₅₅₈ was measured with a UV-Vis spectrophotometer against an appropriate reagent blank. Hyp was quantified based on a calibration curve according to the equation, y=0.1835x−0.0211 (R²=0.9881), where “y” and “x” represent the A₅₅₈ and Hyp concentration (µg/mL). The total Hyp mass was then employed to calculate the total Hyp content, Hyp/protein ratio, and estimated collagen as follows:

$$\text{Total Hyp content (\%)}=\frac{\text{Mass of quantified Hyp (mg)}}{\text{Mass of initial PSC powder (mg)}}\times 100$$

$$\text{Hyp/Proteins ratio (\%)=}\frac{\text{Mass of Hyp (μg/mL)}}{\text{Mass of total soluble proteins (μg/mL)}}\times 100$$

$$\text{Collagen purity (\%)=}\frac{\text{Mass of quantified collagen (Mass of Hyp}\times 7.14)}{\text{Mass of total soluble proteins (mg)}}\times 100$$

The concentration and mass of total soluble proteins were previously determined utilizing the Lowry method. Total collagen mass was estimated employing the conversion factor 7.14, commonly applied to marine-derived collagens. All analyses were performed in triplicate (n=3).

Amino acid profiling by high-performance liquid chromatography

The amino acid composition of the lyophilized PSC was determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with pre-column phenylthiocarbamyl derivatization, following the original procedure of Janssen et al. (1986) as adapted by Mazouzi et al. (2023). Briefly, 10 mg of PSC was hydrolyzed in 300 µL of 6 N HCl under a nitrogen atmosphere at 110°C for 24 h to release amino acids under standard hydrolysis conditions. After cooling to room temperature, the hydrolysates were filtered through 0.22 µm polyvinylidene fluoride membranes and evaporated to dryness under vacuum at 60°C to remove any residual acid. The dried residues were reconstituted in an appropriate volume of solvent A and pre-column derivatized by reaction with phenylisothiocyanate (10% w/v) in pyridine/water/triethylamine (7:1:1, v/v/v) for 40 min at room temperature in the dark, yielding stable phenylthiocarbamyl amino acid derivatives with UV absorbance at 254 nm. Excess reagent was removed before injection by vacuum evaporation.

Chromatography used a YL9100 HPLC system equipped with a YL9120 UV-Vis detector and a Phenomenex Luna C18 column (150×4.6 mm, 5 µm particle size) maintained at 40°C. The binary mobile phase consisted of solvent A: anhydrous sodium acetate buffer, 0.14 mol·L⁻¹, pH 5.9, containing 6% acetonitrile (v/v) and solvent B: acetonitrile/water, 60:40 (v/v), both of HPLC grade. Gradient elution was applied as follows: 0－1 min, 100% A; 1－6 min, linear gradient from 100% to 40% A; 6－8 min, linear gradient from 40% to 0% A; 8－9 min, re-equilibration at 100% A. The total run time was 10 min at a constant flow rate of 0.8 mL·min⁻¹. The injection volume was 20 µL, and the column was equilibrated for 2 min between consecutive injections; detection was at 254 nm.

Amino acids were identified by comparing their chromatographic retention times with those of certified external reference standards (Sigma-Aldrich) analyzed under identical conditions. Absolute quantification used six-point external calibration curves plotted via gravimetric serial dilution at 0.125－4.0 µmol·mL⁻¹. Only calibration functions with a coefficient of determination (R²)≥0.995 were accepted for sample quantification.

To account for the amino acids contributed by the exogenous pepsin introduced during enzymatic extraction, a procedural blank was prepared by subjecting an equal amount of pepsin, at an E/S ratio of 1:20 (w/w), to identical hydrolysis, derivatization, and chromatography conditions. The net amino acid contents attributable exclusively to the byssus-derived collagen fraction were quantified by subtracting the pepsin blank values from the gross PSC measurements. Tryptophan was not determined as it is degraded under the acidic hydrolysis conditions employed. The results are expressed as grams of amino acid per 100 g of total corrected protein on a dry weight basis (g/100 g protein) and are reported as mean±standard deviation (n=3).

Animals and experimental design

All experimental procedures were in strict accordance with institutional guidelines for animal care and use and were approved by the Research Ethics Committee of the Faculty of Natural and Life Sciences, Ahmed Ben Bella University, Oran, Algeria (Approval number: N-991/12/2020). The study followed the ethical principles of the Council for Laboratory Animal Protection and Use, Council of European Communities (1987).

Male Wistar rats (Pasteur Institute of Algiers), aged 5 weeks and weighing 80±7 g, were used. Throughout the study, after a 1-week acclimation period, a control group (C; n=8) received a standard diet (St-diet) consisting of casein, sucrose, corn starch, vegetable oil, cellulose, minerals, and vitamins mixtures; the other group of animals (n=16) was fed the St-diet supplemented with 30% fat and 25% sucrose, constituting the HFHS-diet. At the end of 12 weeks, once the rats reached a body weight of 356±12 g, they were divided into two homogeneous groups (n=8): one untreated group continued on the HFHS diet, and the other treated group was fed the HFHS diet supplemented with 12% dried and finely ground mussel byssus (HFHS-by), for 28 days. This design was intended to model a therapeutic intervention, whereby byssus was introduced after the induction of metabolic dysregulation by the HFHS diet, rather than as a preventive strategy. This inclusion level was selected based on established supplementation protocols for marine co-products and biomass; previous studies utilizing similar inclusion rates (10%－12%) in high-fat+high-sugar rodent models have consistently demonstrated marked hypolipidemic, antihyperglycemic, and anti-inflammatory influence without any evidence of toxicity (Mayer et al., 2019, 2022; Abbate et al., 2021). Food intake (FI) was set at 25 g of diet per rat per day; water was provided ad libitum. The animals were placed in metabolic cages under controlled climatic conditions: 23±3°C, 67±4% humidity, and a 12 h light/dark cycle.

Food consumption for each individual was recorded daily during the four-week experimental period. Body weight was measured on day 1 (before the first feeding) and weekly thereafter until day 28. These data were used to calculate the feed efficiency ratio (FER, %)=[body weight gain (g/rat/d)/food intake (g/rat/d)]×100. This parameter was utilized to evaluate the metabolic efficiency of nutrient utilization and the effects of the HFHS diet and/or on energy (Lee et al., 2015).

Subacute toxicity

A subacute toxicity was studied following the Organisation for Economic Co-operation and Development Guideline 407 to evaluate the safety of oral byssus administration. Rats were fed a diet supplemented with 12% byssus for over 28 days. No signs of toxicity, behavioral abnormalities, or mortality were observed throughout the study. These findings indicate that 12% byssus does not produce any apparent toxic impacts under the experimental conditions employed.

Sample collection and preparation for blood

On day 28, after a 12-h fast, eight rats per group were weighed and anesthetized with an intraperitoneal injection of 3 mL/kg chloral hydrate (PRS Panreac), and blood was collected by cardiac puncture. It was centrifuged at 3,000 g for 15 min at 4°C (Hettich Rotina 38R centrifuge) to obtain serum, of which HbA1c, insulin, lipid profile [total cholesterol, high-density lipoprotein-cholesterol (HDL-C), LDL-C, TGs] were determined, and hepatic enzymes (aspartate aminotransferase and alanine aminotransferase, AST and ALT) were assayed.

Glycemic balance markers

All blood samples were collected under standardized conditions. For measuring fasting blood glucose, animals were starved for 12 h overnight with free access to water. Postprandial glycemia was assessed exactly 2 h after diet administration. Blood samples were collected from the tail vein between 08:00 AM and 10:00 AM to minimize circadian variability. Fasting and postprandial blood glucose levels were assessed weekly over the 120-d experimental period with a validated Vital Check MM1200 glucometer (Tianjin Empecs Medical Device Co., Ltd.) according to the manufacturer’s instructions. HbA1c contents of whole blood samples collected after a 12-h fast using cation-exchange chromatography (Human Diagnostics Worldwide, Ref. 10658) were ascertained at the end of the experiment. Serum insulin levels were determined from fasting blood samples with a commercial enzyme-linked immunosorbent assay (ELISA) kit (A. Menarini Diagnostics, Zenit reagents, Ref. 39818), and serum glucose was quantified utilizing a glucose oxidase-peroxidase enzymatic kit (BioSystems, Ref. 11504).

Lipid profile, cholesterol distribution in lipoproteins, atherogenic ratios, and hepatic biochemical markers

Serum lipids were profiled using enzymatic colorimetric kits supplied by Biolabo SAS: total cholesterol (TC) and HDL-C were measured by the cholesterol oxidase-peroxidase-aminoantipyrine method, TG by the glycerol phosphate oxidase-peroxidase method, and LDL-C by a direct enzymatic approach. Very low density lipoprotein-cholesterol (VLDL-C) was calculated using the following formula, VLDL-C=TG/5, the atherogenic index was calculated as log(TG/HDL-C) (Hermans et al., 2012).

AST and ALT activities were quantified employing the kinetic International Federation of Clinical Chemistry and Laboratory Medicine method without pyridoxal phosphate, with kits supplied by Biomaghreb.

Tissue sample preparation and total protein measurement

Animals were sacrificed, and liver tissues were rapidly excised, washed with ice-cold phosphate-buffered saline (PBS, pH 7.2), blotted dry, weighed, minced, and homogenized: 1 g in 10 mL of PBS. Homogenates were centrifuged at 10,000 g for 10 min at 4°C; supernatant proteins were quantified by the Lowry method (Lowry et al., 1951). Aliquots, 100 µL, were mixed with 1 mL alkaline copper reagent 2% Na₂CO₃ in 0.1 N NaOH mixed with 1% CuSO₄ in potassium-sodium tartrate and incubated at room temperature for 10 min. Then 100 µL of diluted 1 N Folin-Ciocalteu reagent was added, and incubated for 30 min in the dark. The A₇₅₀ was read vs. a buffer blank using a BSA standard curve (0－100 µg/mL). The supernatants were flash-frozen and stored at −80°C.

Redox status

Lipid peroxidation in serum and erythrocytes: Peroxidation of circulating lipids was determined by quantifying thiobarbituric acid reactive substances (Yagi, 1976), with malondialdehyde (MDA) as a marker of oxidative damage. Blood samples were centrifuged at 3,000 g for 10 min to separate the serum and packed red cell volumes. Erythrocytes were washed three times with 0.9% NaCl and subsequently hemolyzed by adding ice-cold distilled water in a 1:4 ratio. For the assay, 200 µL of serum or hemolysate was mixed with 1 mL of 50% trichloroacetic acid (TCA) in 0.1 M HCl and 1 mL of 0.1 M thiobarbituric acid (TBA). After vortexing, the mixture was heated in a water bath at 95°C－100°C for 30 min. After rapid cooling and centrifugation at 5,000 g for 10 min, the A₅₃₂ of the pink MDA-TBA complex was measured with a UV-1800 UV-Vis spectrophotometer (Shimadzu). Concentrations were calculated using the molar extinction coefficient (ε=1,56×10⁵ M⁻¹ cm⁻¹) and normalized to serum proteins (nmol/mL or mg/mL) or hemoglobin contents (nmol/g Hb) for erythrocytes.

Measurement of hepatic lipid peroxidation: Lipid peroxidation was quantified per the procedure described by Elmallah et al. (2017), where MDA serves as an indicator of oxidative damage. Briefly, 200 µL of tissue supernatant was mixed with a reaction solution containing 1 mL of 50% TCA in 0.1 mL HCl, and 1 mL of TBA prepared in 0.1 M HCl. After vortexing, the mixture was heated, cooled, and centrifuged at 5,000 g for 10 min. The A₅₃₂ of the pink chromogen (MDA-TBA complex) obtained was determined. MDA levels were calculated and expressed as nmol MDA per mg of protein (Elmallah et al., 2017).

Superoxide dismutase (SOD) assay: The serum, hepatic tissue, and erythrocytes’ SOD activity was determined using the nitro blue tetrazolium (NBT) photoreduction inhibition assay. While serum and liver homogenate supernatants 200 µL were processed directly, erythrocytes underwent a preliminary preparation step involving three washes with 0.9% NaCl and hemolysis in ice-cold distilled water in a ratio of 1:4 to minimize hemoglobin interference. The 1.5 mL reaction mix consisted of the sample, 100 mM Tris-HCl buffer (pH 7.8), 6 mM ethylenediaminetetraacetic acid, 75 µM NBT, and 2 µM riboflavin. Following a 15-min exposure to a calibrated light source, the A₅₆₀ was recorded on a UV-1800 UV-Vis spectrophotometer. One unit (U) of SOD activity was defined as the amount of enzyme required to inhibit NBT photoreduction by 50%. The results were normalized to total protein (U/mg) for hepatic tissue and serum and to hemoglobin content (U/g Hb) for erythrocytes (Apak et al., 2022).

Catalase assay: Catalase activity in the serum, erythrocytes, and hepatic tissues was determined following the procedure described by Cuéllar-Cruz et al. (2009), with minor adjustments for each matrix. Briefly, 50 µL of each sample tissue supernatant, serum, or diluted hemolysate was added to 3 mL of H₂O₂ prepared in 50 mM phosphate buffer (pH 7.0). H₂O₂ decomposition was monitored spectrophotometrically by measuring the ΔA240 nm. Optical density was recorded for over 2 min at 30 s intervals. The reaction rate was calculated based on H₂O₂ disappearance and expressed as µmol of H₂O₂ decomposed per min per mg of protein (µmol/min/mg protein) for tissue and serum or per gram of hemoglobin (µmol/min/g Hb) for erythrocytes (Cuéllar-Cruz et al., 2009).

Glutathione (GSH) reductase assay: Reduced GSH levels in serum, erythrocytes, and hepatic tissues were determined employing a 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB)-based colorimetric method per the methodological recommendations described by Tipple and Rogers (2012). Briefly, an aliquot of tissue supernatant 1.0 mL was mixed with 1.0 mL of 4% sulfosalicylic acid and incubated for 1 h at 4°C to precipitate proteins. After centrifugation, 0.4 mL of the acidic supernatant was combined with 2.2 mL of 0.1 M phosphate buffer (pH 7.4) and 0.4 mL of 4 mg/mL DTNB solution. The A₄₁₂ of the resulting yellow chromogen was immediately measured using a HumaReader HS Microtiter Plate Reader (Molecular Devices). GSH was quantified based on a standard curve and expressed as nmol GSH per mg of protein (mg protein) for tissue and serum or nmol GSH per gram of hemoglobin (g Hb) for erythrocytes (Tipple and Rogers, 2012).

Paraoxonase-1 (PON1) arylesterase assay: The arylesterase activity of PON1 was determined spectrophotometrically per the method of Bełtowski et al. (2002), with phenyl acetate as the substrate. The reaction mixture, 3 mL, consisted of 20 mM Tris-HCl buffer (pH 8.0) supplemented with 1 mM CaCl₂ and 1 mM phenyl acetate. The reaction was initiated by adding 5 µL of diluted serum, and the hydrolysis of phenyl acetate into phenol was monitored based on the ΔA₂₇₀ for 2 min with a UV-1800 UV-Vis spectrophotometer. Enzymatic activity was calculated utilizing the molar extinction coefficient of phenol (ε=1,310 M⁻¹ cm⁻¹). One U of activity was defined as the amount of enzyme required to hydrolyze 1 µmol of phenyl acetate per min. The results were normalized to the total protein concentration and expressed as U/mg protein (Bełtowski et al., 2002).

Assessment of inflammatory markers

Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6): Levels of pro-inflammatory cytokines, specifically TNF-α and IL-6, in serum and hepatic tissues were determined using commercial ELISA kits (Sigma-Aldrich) per the manufacturer’s instructions. Briefly, 100 µL aliquots of serum or hepatic supernatants were added to and incubated with specific detection antibody-coated microtiter plates. Following washings and incubation with horseradish peroxidase-conjugated secondary antibodies, the substrate was added, and the reaction was terminated with a stop solution. A₄₅₀ was measured with a HumaReader HS microplate reader (HUMAN). Standard curves were constructed utilizing serial dilutions of recombinant TNF-α and IL-6 standards, and samples were quantified by interpolation. The results were normalized to the total protein content and expressed as pg/mg protein.

C-reactive protein (CRP): The serum CRP concentrations were determined using a latex-based immunological agglutination method with a CRP Latex Kit (Cypress Diagnostics). Briefly, 50 µL of undiluted serum was mixed with one drop of a latex particle suspension sensitized with anti-human CRP antibodies. After 2 min of homogenization on a test card, visible agglutination indicated CRPs above the detection threshold of 6 mg/L, equivalent to the positive control. Positive samples were semiquantitatively analyzed using serial dilutions 1/2, 1/4, and 1/8 in 0.9% normal saline to determine the final titer. Notably, this method is semi-quantitative and lacks the quantitative analytical precision of rat-specific ELISA-based assays, representing a limitation of the present study. Therefore, CRP data must be interpreted as indicative of systemic inflammation trends rather than as accurate and absolute quantitative values. Future investigations may employ validated rat-specific quantitative ELISA methods to provide a more accurate and reproducible inflammatory profile.

Statistical analysis

Data are expressed as mean±standard error of the mean for biological parameters (n=8 per group) and as mean±standard deviation for nutritional composition (n=3 determinations). Data normality was assessed using the Shapiro-Wilk test. Statistical analyses were performed using SPSS software version 26.0 (IBM Corp.). Inter-group differences were ascertained by one-way analysis of variance, followed by Tukey’s honestly significant difference post hoc test for multiple comparisons. Variations were considered statistically significant at P<0.05.

RESULTS AND DISCUSSION

HFHS diet induced metabolic alterations in Wistar rats, predisposing them to diabetes, characterized by hyperglycemia, dyslipidemia, and hepatic steatosis with underlying oxidative stress and chronic inflammation (Panchal et al., 2011; Lian et al., 2020), which enhance the risk of severe cardiovascular and hepatic complications. This effect has prompted sustained efforts to investigate the preventive potential of novel natural curatives that could be utilized as functional ingredients against diet-induced hyperglycemia and hyperlipidemia.

In this context, the therapeutic efficacy observed with 12% byssus supplementation aligns with research on marine co-products and biomass. For instance, the inclusion of ingredients derived from marine organisms such as Tisochrysis lutea or Diacronema lutheri at the same 12% rate markedly improves lipid profiles, reduces HbA1c levels, and mitigates systemic inflammation in HFHS-fed rats (Mayer et al., 2019, 2022). Our findings corroborate that this specific dosage of 10%－12% represents a functional threshold for marine bioresources, presumably associated with sufficient concentrations of bioactive peptides and DOPA residues capable of counteracting diet-induced metabolic alterations. This outcome suggests that 12% constitutes an effective and biologically active dose for the valorization of mussel co-products as functional ingredients. However, since dose-responses were not compared, this dosage must not be definitively considered as optimal, and further studies need to test a range of concentrations to identify the most effective dose.

Nutritional composition of mussel byssus

The biochemical composition of 1 g of M. galloprovincialis byssus powder was determined, and the results are expressed as percentages of the total mass (Table 1). The biochemical profile of byssus confirmed its protein-rich nature; however, the proportion determined (70.82±0.51%) was lower than the 84.37% reported by Lassoued et al. (2016). Such variations may result from differences in environmental conditions, sampling seasonality, or analytical methods (Meloni et al., 2022). The lipid content (2.90±1.12%) was higher than published values (1.94%), which may reflect physiological adaptations or local conditions influencing structural flexibility, interfiber cohesion, and hydration resistance (Fokina et al., 2014; Dernekbasi et al., 2015). Similarly, the carbohydrate fraction (3.28±0.96%) exceeded earlier measurements (0.87%), suggesting a greater abundance of structural glycoproteins or polysaccharides crucial for maintaining its mechanical and adhesive properties (Choi et al., 2020). The ash content (18.80±0.79%) indicated a substantial incorporation of inorganic elements, likely derived from the environment and substrates; this value was markedly greater than the 9.67% reported (Rodríguez et al., 2017). The residual moisture content (2.20±1.34%) sharply contrasted with the 67.07% reported by Lassoued et al. (2016), likely due to a more intensive dehydration protocol employed in our study that concentrated the remaining components (Lo Presti and Frieß, 2021). Overall, these differences underscore the combined influence of ecological, biological, and methodological factors on the biochemical composition of M. galloprovincialis byssus.

Table 1.

Characterization of the nutritional composition of mediterranean mussel (Mytilus galloprovincialis) byssus

Components (%)	Results
Proteins	70.82±0.51
Total lipids	2.90±1.12
Carbohydrates	3.28±0.96
Ash content	18.80±0.79
Moisture	2.20±1.34

Values are presented as a percentage of dry weight and represent the mean±standard deviation of triplicates (n=3).

Extraction kinetics and optimization of hydrolysis duration

To maximize collagen recovery from the dense M. galloprovincialis byssus matrix, pepsin digestion was successfully implemented and optimized by evaluating the influence of treatment duration 4 h vs. 24 h at a fixed E/S ratio of 1:20 (w/w). Pepsin is highly advantageous for this bioprocess due to its selective activity; while generally it is indiscriminate in digesting non-collagenous proteins, the native triple-helical domain of collagen is entirely resistant to enzymatic cleavage. Pepsin specifically targets and cleaves crosslinked telopeptide regions without compromising the structural integrity of the central triple helix through this limited proteolytic mechanism (Qian et al., 2016; Rodríguez et al., 2017).

In our study, evaluation of enzymatic kinetics revealed a pronounced temporal dependency. The shorter incubation period of 4 h limited the extraction yield to 18.61±1.57%, corresponding to a PSC recovery of 2.29 g, indicating extensively incomplete matrix hydrolysis. Conversely, extending the duration to 24 h markedly enhanced process efficiency, achieving a remarkable yield of 29.75±1.96%, equivalent to 3.57 g of lyophilized PSC from the initial 12 g of matrix (Fig. 1). From a structural perspective, the marine bivalve byssus represents a rigid, acellular matrix, extensively crosslinked and stabilized by a quinone-tanning process involving DOPA residues (Rzepecki et al., 1992; Waite, 2017). Such a dense molecular architecture is presumed to impose severe steric hindrance, which may initially restrict the accessibility of cleavage sites to pepsin.

Fig. 1.

Yield of pepsin-solubilized collagen extracted from raw Mytilus galloprovincialis byssus powder after enzymatic hydrolysis for two durations: 4 and 24 h, using a constant substrate: pepsin ratio of 1:20. Data are presented as the mean±standard deviation of triplicate experiments (n=3). Various letters indicate statistically significant differences (P<0.05) between yields under separate extraction conditions.

A 4 h incubation period (yield <20%) may be insufficient to permit effective diffusion of a bulky enzyme into the core of pre-treated byssus fibrils. Therefore, extending the extraction time to 24 h appears to constitute an indispensable condition for improving kinetic behavior, which contrasts with certain observations in the literature, where digestion parameters primarily affected the concentrations of isolated amino acids rather than the total protein mass (Vallejos et al., 2014; Luo et al., 2017; Rodríguez et al., 2017). In our optimized system, the 24 h framework is hypothesized to promote matrix swelling within the acidic medium and to provide a temporal window required for the enzyme to overcome the steric hindrance imposed by the quinone-tanned network. Such progressive cleavage ruptures inter- and intra-molecular cross-links (Kim, 2013), potentially allowing structural breakdown to manifest macroscopically as a significantly higher recovery without degrading the central triple-helical structure. Consequently, these outcomes suggest that a 24 h framework at an E/S ratio of 1:20 (w/w) may represent a favorable kinetic compromise to overcome the byssus’ structural resistance and maximize PSC recovery, although this interpretation remains hypothetical in the absence of any direct structural analyses (e.g., microscopy or matrix swelling measurements), and a broader range of durations was not tested to establish a true optimum.

To accurately contextualize the efficacy of this kinetic strategy, these outcomes must be evaluated against earlier attempts at isolating collagen from identical or alternative marine matrices with comparable densities. Prior extractions of mussel byssus reported a maximum yield of only 7.6% on a Hyp basis (Rodríguez et al., 2017), highlighting the superior performance of our optimized pre-treatment and hydrolysis loop. Furthermore, the competitive advantage of the 29.75% yield achieved is evidenced when comparing it to alternative, less complex marine matrices. It remarkably surpasses the recovery efficiencies for specific teleost and elasmobranch structures, including Aluterus monoceros (8.4%) and Diodon holocanthus (19.5%), while remaining highly comparable to those documented for Thunnus albacares (27.1%) and Carcharhinus limbatus (20.9%) (Woo et al., 2008; Ahmad and Benjakul, 2010; Kittiphattanabawon et al., 2010; Huang et al., 2011). These stark variations underscore that our 24 h framework achieves a favorable equilibrium between mass transport diffusion and selective macromolecular solubilization, providing a highly efficient and reproducible methodology for processing dense invertebrate-derived biomaterials.

Compositional analysis of isolated PSC

The composition of isolated PSC was quantitatively analyzed to establish the procedural efficiency of purification and dialysis (Table 2). Protein content was determined using the Lowry method (Lowry et al., 1951), with BSA as an external standard and calibration curve: y=0.3851x+0.0759; R²=0.983. The mean A₇₅₀ recorded for the working solution with an initial concentration of 1 mg·mL⁻¹ was 0.429, corresponding to a soluble protein concentration of 0.916 mg·mL⁻¹. The total protein content of the lyophilized PSC was thus estimated to be 91.59% (915.9 mg·g⁻¹ dry weight; Table 2), reflecting a high purity of the isolated fraction protein.

Table 2.

Compositional analysis and purity indices of isolated mediterranean mussel (Mytilus galloprovincialis) pepsin-solubilized collagen

Parameter (%)	Value
Total protein (Lowry method)	91.59±2.29
Hydroxyproline (colorimetric assay)	12.48±2.47
Estimated collagen (Hyp×7.14)	89.11±1.38
Collagen-to-protein purity index	97.29±1.13

Values are presented as mean±standard deviation of triplicate experiments (n=3).

Hyp, hydroxyproline.

Collagen-specific quantification was via the colorimetric determination of Hyp, a reliable biochemical marker of collagen due to its near-exclusive occurrence in this family of proteins. The Hyp content of the PSC powder was 12.48% (124.8 mg·g⁻¹ dry weight; Table 2). Using the widely adopted marine collagen conversion factor of 7.14 (Hyp×7.14), the estimated absolute collagen level was 89.11% (891.1 mg·g⁻¹ dry weight; Table 2). The collagen purity index, expressed as the collagen-to-total-protein ratio, reached 97.29%, indicating that the collagen was highly enriched in the isolated fraction, with non-collagenous protein impurities accounting for only 2.71% (Table 2).

These findings agree with the values reported for collagen extracted from marine invertebrates. Vallejos et al. (2014) reported a collagen content of 69 mg·g⁻¹ protein with a Hyp level of 1.8 mg·g⁻¹ protein for PSC extracted from the byssus of Chilean mussels (Mytilus chilensis) using a pepsin-aided extraction protocol. Notably, their Hyp concentration and overall macromolecular recovery were remarkably lower than those achieved in this study (Vallejos et al., 2014). Collagenous fragments isolated from the byssal threads of Mytilus edulis were also characterized based on Hyp and glycine levels of ∼4% and 35%, respectively; compositional features were consistent with structural collagen (Qin and Waite, 1995). In M. galloprovincialis, biochemical characterization of byssal threads revealed a proximate composition dominated by proteins, with total nitrogen reaching 13.50±0.24% dry weight, confirming the proteinaceous and collagen-enriched nature of this marine biopolymer (Hagenau et al., 2009).

Furthermore, lyophilized collagen prepared from the byproducts of marine fish processing has yielded protein contents >90% dry weight, with collagen representing ∼85% of total protein (Zarubin et al., 2024), values comparable to those obtained in the present study: 91.59% total protein; 97.29% collagen purity. The high purity index achieved here definitively confirms the effectiveness and selectivity of the salting-out precipitation and dialysis steps of collagen concentrating while eliminating non-collagenous matrices from the mussel byssus as a substrate.

Amino acid composition of the PSC

The amino acid profile of the PSC isolated from M. galloprovincialis byssus, determined by RP-HPLC, is graphically represented as percentages of total protein (g/100 g protein) in Fig. 2, while the raw chromatographic separation and representative amino acid peaks (mV.s vs. min) are illustrated in Fig. 3. It displayed well-resolved, symmetrical peaks for all amino acid residues detected, with glycine producing the most intense signal, consistent with its predominant relative abundance among all quantified residues. Additional unidentified peaks were not observed, supporting the reliability of the analysis and purity of the derivatized PSC hydrolysate. Glycine was identified as the most abundant residue (31.24±0.24 g/100 g protein), followed by proline (12.78±0.31 g/100 g protein), alanine (8.61±0.32 g/100 g protein), arginine (7.37±0.19 g/100 g protein), phenylalanine (2.34±0.14 g/100 g protein), and tyrosine (0.81±0.17 g/100 g protein) (Fig. 2). Hyp, quantified independently by colorimetric assay, accounted for 12.48 g/100 g protein, yielding a combined imino acid content (proline+Hyp) of 25.26 g/100 g protein.

Fig. 2.

Amino acid profile of purified pepsin-solubilized collagen from Mytilus galloprovincialis byssus obtained by reverse-phase high-performance liquid chromatography (n=3, mean±standard deviation).

Fig. 3.

Representative reverse-phase high-performance liquid chromatography chromatogram of amino acids derivatized from Mytilus galloprovincialis pepsin-solubilized collagen, using phenylthiocarbamyl, showing resolution peaks mV.s as a function of retention time (min). The chromatogram is representative of three independent replications (n=3). Arg, arginine; Pro, proline; Phe, phenylalanine; Tyr, tyrosine; Ala, alanine; Gly, glycine.

The predominance of glycine is universally recognized as the structural hallmark of fibrillar collagen. As the most abundant amino acid of marine collagen, glycine consistently accounts for >30% of all residues. Variations in the overall amino acid profile, particularly regarding the Hyp content, are generally reported to influence crucial collagen properties, such as matrix rigidity, conformational stability, and thermal denaturation temperature. The glycine levels recorded in this study (31.24±0.24 g/100 g) substantially exceeded the 14.3 g/100 g reported by Vallejos et al. (2014) for PSC from M. chilensis byssus while remaining fully consistent with the values documented for PSC from other marine sources. For instance, in the PSCs isolated from the swim bladders of four fish species, glycine consistently represented the greatest proportion of total residues at approximately one-third (289－311/1,000 residues), followed by proline (114－136/1,000 residues) and alanine (108－114/1,000 residues), further confirming the Gly-X-Y repeat sequence as the fundamental structural basis of the collagen triple helix (Vallejos et al., 2014).

The combined imino acid content (25.26 g/100 g) recorded in this study exceeded the 20% threshold considered diagnostic of type I-III fibrillar collagens and substantially surpassed the proline content of 6.8 g/100 g reported for M. chilensis (Vallejos et al., 2014). The concentration of imino acids (proline and Hyp) was typically elevated in PSC than in acid-soluble collagen fractions. This outcome reflects the selective action of pepsin, which preferentially cleaves non-helical telopeptides while liberating intact triple-helical domains enriched in imino residues. These findings are remarkably consistent with the landmark investigation by Qin and Waite (1995), who demonstrated that pepsin-resistant collagen fragments (Col-P) isolated from M. edulis byssal threads display a characteristic collagenous composition, containing ∼35% glycine and nearly 20% proline plus 4-trans-Hyp structured as tandem Gly-X-Y triplet repeats. The values obtained here for M. galloprovincialis PSC provide robust evidence for the structural identity and enhanced purity of the isolated fraction (Qin and Waite, 1995; Dong and Dai, 2022; Oslan et al., 2022).

Furthermore, the alanine (8.61±0.32 g/100 g), arginine (7.37±0.19 g/100 g), and phenylalanine (2.34±0.14 g/100 g) levels were markedly greater than those reported by Vallejos et al. (2014) for M. chilensis PSC (3.9, 5.8, and 1.9 g/100 g, respectively). While marine collagen is generally characterized by elevated concentrations of alanine, proline, Hyp, and glutamic acid, it typically exhibits low levels of cysteine, tyrosine, histidine, and methionine, depending on species and tissue of origin. The systematically higher amino acid contents observed in this study relative to those reported in the literature were consistent with the extensive purification achieved by our optimized salting-out-based precipitation and dialysis steps, which selectively concentrated the structural collagenous fraction (Blanco et al., 2017; Ge et al., 2022).

The tyrosine content (0.81±0.17 g/100 g) is of particular biochemical relevance in the context of the mussel byssus. These residues serve as obligate precursors for the post-translational biosynthesis of DOPA, the catecholic amino acid responsible for the exceptional adhesive and cross-linking properties of byssal foot proteins (Qin and Waite, 1995). Tyrosine, histidine, and cysteine were consistently reduced in pristine marine structural collagen, a compositional feature that distinguishes it from non-collagenous proteins. Crucially, the complete absence of cysteine (Fig. 3) further corroborates the collagenous purity of the isolated fraction, as low or undetectable cysteine levels are a definitive characteristic of structural fibrillar collagens (Lim et al., 2019; Salvatore et al., 2020).

Together, the amino acid profile of the PSC isolated from M. galloprovincialis byssus, dominated by glycine, is enriched with substantial imino acid, alanine, and arginine fractions, displaying baseline tyrosine levels, and lacking cysteine. It is fully consistent with a high-purity type I marine invertebrate fibrillar collagen, confirming the structural integrity and definitive collagenous identity of the isolated macromolecule.

Growth parameters, FER, and FI

By day 28, chronic consumption of the HFHS diet induced a profound metabolic shift in the experimental models compared to the control group (Table 3). Despite developing pronounced hypophagia, the untreated HFHS rats paradoxically exhibited an approximately twofold increase in their total body WG (P<0.01) and a marked elevation in their FER (P<0.001). Such striking divergence, where lower FI co-occurs with accelerated WG, reflects a severe diet-induced disruption in energy homeostasis, typically driven by higher caloric density and metabolic inflexibility shifting the system toward adipose storage rather than energetic expenditure (Moreno-Fernández et al., 2018; Garcia et al., 2019). Dietary intervention with Mediterranean mussel byssus proved effective in reversing this trajectory of profound metabolic dysregulation. In the HFHS-by group, excessive WG (P<0.01) and elevated FER (P<0.001) were markedly attenuated compared to the untreated HFHS group (Table 3), while voluntary FI was partially normalized. This robust improvement in energy efficiency, reflected by a lower FER despite greater FI, aligns with reports on marine peptides with bioactivity, which modulate lipid turnover and restrict hepatic lipoaccumulation in dyslipidemic models (Wang et al., 2008; Vijayan et al., 2022). Although the final growth parameters of the treated rats remained at levels intermediate between those of the control and HFHS groups, the overall pattern demonstrated a substantial therapeutic restoration of metabolic balance. Given the rich collagenous nature of the mussel byssus matrix, these impacts are likely sustained by enhanced lipid catabolism in peripheral tissues, offering a viable natural strategy to counteract long-term obesity-related complications (Wang et al., 2008; Woo et al., 2018).

Table 3.

Impact of Mytilus galloprovincialis byssus consumption on overall food intake, body weight gain, and feed efficiency ratio in Wistar rats

Parameter	Control	HFHS	HFHS-by
Initial body weight (day 0; g/rat)	223.01±15.03	359.10±11.06***	353.11±12.55***ns
Final body weight (day 28; g/rat)	251.06±16.01	423.30±17.05***	386.10±12.03***ns
Body weight gain (g/rat/d)	1.03±0.04	2.28±0.23**	1.17±0.25ns##
Food intake (g/rat/d)	22.03±2.10	18.10±1.65ns	21.07±1.74ns
Feed efficiency ratio (%)	4.55±0.05	12.67±0.09***	5.58±0.18***###

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

*P<0.05, **P<0.01, and ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01, and ^###P<0.001 vs. HFHS group. ns, not significant.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus.

Effect of mussel byssus on serum lipid profile

A comprehensive analysis of the lipid profile revealed marked metabolic alterations following dietary interventions (Table 4). Rats fed the HFHS diet developed pronounced dyslipidemia compared to the controls, as evidenced by sharp elevations in TC, TGs, LDL-C, and VLDL-C (P<0.001), which was concomitant with a profound reduction in cardioprotective HDL-C (P<0.001). These severe lipid disturbances culminated in a dramatically elevated TG/HDL-C ratio (P<0.001), confirming elevated atherogenic risk. Such a pathological lipid signature is highly characteristic of insulin resistance, lipid overproduction, and diet-induced metabolic disorders, aligning with well-established models of chronic high-calorie intake (Nakhjavani et al., 2020; Qahremani et al., 2023).

Table 4.

Effects of Mytilus galloprovincialis byssus on the serum lipid profiles of HFHS diet-induced hyperlipidemic and hyperglycemic rats

Parameter	Control	HFHS	HFHS-by
Total cholesterol (mg/dL)	66.01±0.24	110.03±0.37***	82.96±0.28***###
Triglycerides (mg/dL)	92.04±0.25	177.10±0.16***	134.07±0.24***###
HDL-C (mg/dL)	29.83±0.54	18.13±0.59***	25.22±0.73***###
LDL-C (mg/dL)	18.37±0.29	57.12±0.79***	31.02±0.85***###
VLDL-C (mg/dL)	18.45±0.35	35.38±0.62***	26.85±0.95***###
Triglycerides/HDL-C	3.08±0.32	9.76±0.81***	5.31±0.34*###

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

*P<0.05 and ***P<0.001 vs. control group; ^###P<0.001 vs. HFHS group.

HFHS, high-fat, high-sucrose diet-fed group; Control, control group fed a standard diet; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; VLDL-C, very low density lipoprotein-cholesterol.

Therapeutic intervention with the Mediterranean mussel byssus was highly effective in counteracting these lipid disruptions. Compared to the untreated HFHS group, the HFHS-by cohort exhibited a robust reduction in circulating TC, TG, and LDL-C levels (P<0.001), alongside a remarkable recovery of serum HDL-C (P<0.001; Table 4). Furthermore, composite atherogenic indices were favorably modulated, highlighted by marked declines in VLDL-C concentrations (P<0.001) and the TG/HDL-C ratio (P<0.001). The clinical relevance of utilizing composite ratios like TG/HDL-C lies in their capacity to integrate multiple lipid fractions, providing sensitive and reliable indicators of cardiovascular risk and systemic metabolic imbalances (Pereira, 2012). The magnitude of these lipid level improvements prompts a consideration of the underlying mechanisms driving the hypolipidemic potential of the mussel byssus matrix. These effects are likely driven by its unique biochemical composition, particularly its abundance of specific amino acids, such as arginine and glycine (Lassoued et al., 2016). Dietary arginine effectively lowers plasma cholesterol and TG levels in obese rodent models by enhancing insulin sensitivity and overall metabolic expenditure (Wu et al., 2009; Rom et al., 2018). Moreover, the structural matrix of byssus contains unique bioactives, including precollagens D, P, and NG, Hyp, and LDOPA, which may exert synergistic therapeutic effects. For instance, DOPA confers potent intrinsic antioxidant properties that could plausibly suppress oxidative stress and restrict lipid peroxidation, potentially reducing circulating TG and cholesterol levels observed here. Consistent with the reported mechanisms, it is plausible that the bioactives present in byssus could attenuate hepatic lipid synthesis and promote fatty acid β-oxidation. However, these mechanistic interpretations remain hypothetical without direct molecular evidence and require validation in future studies incorporating gene expression and protein quantification studies (Woo et al., 2018; Tometsuka et al., 2021; Souza-Tavares et al., 2023). These findings corroborate recent reports demonstrating that marine collagen-derived bioactive peptides efficiently modulate hepatic lipogenesis and enhance peripheral lipid turnover, reinforcing the commercial value of byssus as a promising marine bioresource (Wang et al., 2008; Carrera et al., 2023). Notably, despite robust improvements observed in the treated cohort, the 28-d intervention achieved a substantial but only partial restoration of lipid homeostasis. When compared directly to the healthy control group, the HFHS-by rats still displayed intermediate-moderate lipid abnormalities, maintaining higher baseline levels of TC, TG, and atherogenic lipoproteins (P<0.001; Table 4). This trend indicates that while mussel byssus exerts a powerful corrective effect on lipid metabolism, a longer experimental duration or a higher dosage may be required to fully reverse chronic diet-induced dyslipidemia.

Effect on glucose, insulin, HbA1c, and postprandial glycemia rates

In the HFHS rodent model, insulin resistance and impaired glucose homeostasis typically provoke a severe mismatch between pancreatic insulin secretion and systemic glycemic load. This cascade causes persistent fasting and postprandial hyperglycemia, along with elevated glycated hemoglobin (HbA1c) within the metabolic syndrome-associated clinical cluster (Moreno-Fernández et al., 2018). Tracking data confirmed such a pathological progression, as both fasting and postprandial blood glucose levels exhibited a profound, sustained escalation in the untreated HFHS group from the study onset through day 28 compared to the healthy controls (Fig. 4). Such a chronic hyperglycemic state was accompanied by a non-significant declining trend in serum insulin levels and a marked accumulation of HbA1c (P<0.001; Table 5), signaling progressive pancreatic β-cell exhaustion and long-term glycemic failure induced by the high-calorie dietary regimen.

Fig. 4.

Weekly measurements of postprandial blood glucose (A) and fasting blood glucose (B) in Wistar rats of the control group fed a standard diet (Control), high-fat, high-sucrose diet-fed (HFHS), and high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground Mytilus galloprovincialis byssus (HFHS-by) groups (n=8 per group). Data are presented as the mean±standard error of the mean for each week. Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test. *P<0.05 vs. control group; ^#P<0.05 vs. HFHS group.

Table 5.

Effects of Mytilus galloprovincialis byssus on circulating insulin and HbA1c levels

Parameter	Control	HFHS	HFHS-by
Serum insulin (µIU/mL)	14.01±3.30	9.09±1.09ns	11.79±0.78ns
HbA1c (%)	3.10±0.29	4.93±0.31***	4.20±0.12*ns

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

*P<0.05 and ***P<0.001 vs. control group. ns, not significant.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus.

Dietary supplementation with Mediterranean mussel byssus effectively reversed these glycemic disruptions.

Fasting and postprandial glucose levels were substantially and parallely reduced in the HFHS-by-cohort compared to the untreated HFHS group (P<0.05; Fig. 4). This systemic recovery was further supported by a declining trend in HbA1c percentages, though without reaching statistical significance compared to the untreated HFHS group, (P<0.05; Table 5), while insulin levels showed no significant change.

Notably, in this regard, unlike humans, whose erythrocyte lifespan averages ∼120 days, the erythrocyte turnover rate in rats is significantly accelerated to 50－60 days (Beltran Del Rio et al., 2016). This compressed lifespan suggests that rodent HbA1c levels reflect a much shorter window for glycemic exposure, rendering a relevant and statistically detectable reduction in HbA1c over the 28-d intervention period physiologically plausible in this animal model.

The marked glycemic improvements in the treated group may be mediated by the bioactivity of marine-derived structural proteins and bioactive peptides inherent to the byssus matrix. Based on the literature, we hypothesize that these biopeptides could exert antidiabetic effects by inhibiting critical carbohydrate-digesting enzymes, such as α-amylase and α-glucosidase, thereby potentially flattening postprandial glucose fluctuations by restricting intestinal glucose absorption (Gunathilaka et al., 2022).

Furthermore, these components may modulate incretin metabolism via the competitive inhibition of dipeptidyl peptidase-IV, a mechanism known to preserve endogenous insulin secretion and protect the pancreatic architecture from chronic metabolic stress (Xia et al., 2017; Chen et al., 2024). Such protection could be further supported by a restoration of circulating insulin levels in the HFHS-by group, which may facilitate downstream hepatic glycogen storage and attenuate ectopic lipid deposition within peripheral tissues (Zhou et al., 2017).

Additionally, the rich functional amino acid profile of byssus, specifically arginine, may act synergistically to enhance insulin sensitivity and skeletal muscle glucose uptake via nitric oxide-dependent pathways, mirroring the metabolic improvements in conventional insulin-sensitizing strategies (Jobgen et al., 2009; Choi et al., 2017).

While based on structural analogies with other marine collagen matrices, these enzymatic and receptor-level mechanisms are highly plausible; they remain hypothetical within the scope of this study, as direct molecular or enzyme kinetic assays were not performed. Future investigations involving Western blots and enzyme inhibition are required to definitively map these specific intracellular pathways.

Crucially, this metabolic correction was notably partial. By the conclusion of the study, the HFHS-by group maintained intermediate baseline values, with HbA1c remaining moderately higher, than those of the healthy controls (P<0.05; Table 5) while insulin levels showed no significant difference.

This target-tissue lag suggests that while mussel byssus initiates a robust amelioration in systemic glucose homeostasis, full cell-level restoration may require a prolonged therapeutic window to completely clear the metabolic footprint of chronic HFHS exposure.

Effects on the contents of hepatic function biochemical markers

Evaluation of serum transaminase activities across experimental groups revealed pronounced hepatic alterations induced by the hypercaloric diet (Table 6). Rats fed the HFHS regimen exhibited marked elevations in ALT and AST activities, with levels remarkably exceeding those of healthy controls (P<0.001). Such substantial leakage of intracellular enzymes into circulation serves as a definitive indicator of severe hepatocellular injury and compromised membrane integrity.

Table 6.

Hepatic enzyme parameters

Parameter	Control	HFHS	HFHS-by
ALT (IU/L)	91.21±1.66	149.26±1.40***	123.49±0.30***###
AST (IU/L)	54.33±1.45	139.12±0.39***	96.27±0.33***###
Total protein (mg·g—1 wet tissue)	7.26±0.59	6.02±0.44ns	6.93±0.46ns

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

***P<0.001 vs. control group; ^###P<0.001 vs. HFHS group. ns, not significant.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

These findings are fully consistent with the well-documented deleterious impacts of HFHS diets on hepatic structure. Excessive systemic lipid influx and ectopic TG accumulation trigger lipotoxicity, local oxidative stress, and subsequent hepatocytotoxicity (Klobučar Majanović et al., 2021). Furthermore, this severe enzymatic leakage reflects the bidirectional relationship between dysregulated circulating lipoproteins, specifically elevated LDL-C and depleted HDL-C, and a deterioration in liver structural integrity (Deb et al., 2018). This pathological interaction is often exacerbated by an elevated TG/HDL-C ratio, which correlates positively with insulin resistance and drives downstream de novo lipogenesis, perpetuating a self-reinforcing cascade of progressive hepatic deterioration (Son et al., 2021).

Dietary intervention with Mediterranean mussel byssus significantly counteracted this hepatocellular damage. A robust attenuation of serum ALT and AST was seen in the HFHS-by group compared to the untreated HFHS cohort (P<0.001; Table 6), underscoring the strong hepatoprotective capacity of the marine matrix. This finding aligns with literature demonstrating that marine organism-derived bioactive peptides, particularly those sourced from structural collagenous frameworks, can effectively preserve membrane stability, limit hepatocyte leakage, and alleviate diet-induced liver injury (Lin et al., 2012; Han et al., 2015). Beyond transaminase modulation, the liver’s biosynthetic capacity was evaluated through hepatic total protein levels. Chronic HFHS exposure did not significantly affect total protein content compared to the control group (P>0.05), whereas byssus supplementation induced a notable restoration, reversing tissue protein concentrations to control baseline levels, with no significant variations between the HFHS-by group and either the control or untreated HFHS groups (P>0.05). Such an upward trend in the treated group suggests preservation or optimization of hepatic translation machinery, consistent with reports showing that marine collagen peptides can enhance structural protein biosynthesis and bolster overall synthetic hepatic health in metabolic stress models (Wei and Guo, 2022). Together, these findings are consistent with the hypothesis that bioactives derived from mussel byssus may act synergistically to protect hepatic structure and function, underscoring their potential therapeutic relevance in the management of HFHS-induced metabolic liver dysfunction, although this mechanism was not directly tested here.

Effect of mussel byssus on oxidative stress and antioxidant enzyme activities

At the core of hepatocellular damage and systemic metabolic syndrome lies a common pathogenic driver: chronic, diet-induced oxidative stress. Preclinical and clinical evidence establishes that chronic hyperglycemia and hyperlipidemia-induced oxidative stress is a primary driver of diabetic complications and dyslipidemia-associated metabolic instability. Such a state occurs when the excessive generation of ROS outpaces endogenous antioxidant defense networks, causing oxidative damage to essential biomolecules (Sifuentes-Franco et al., 2018).

In this study, chronic exposure to the HFHS diet profoundly impaired systemic and organ-specific oxidative status, characterized by a depletion of key antioxidant enzymes alongside a severe reduction in PON1 arylesterase activity. Dietary intervention with Mediterranean mussel byssus structurally reinforced and partially normalized these redox defense systems across circulating and tissue compartments.

The measured systemic antioxidant profiles of serum and erythrocytes demonstrated distinct strategies involving enzymatic modulation under nutritional stress (Table 7 and 8). In untreated HFHS rats, serum and erythrocytic SOD activities exhibited a moderate adaptive induction, reflecting an endogenous compensatory mechanism aimed at mitigating the initial burst of superoxide radicals generated by metabolic overload. Strikingly, dietary supplementation with mussel byssus profoundly upregulated this defense line, with the SOD of the serum and erythrocytes in the HFHS-by cohort hyper-activating intensely, elevating these defenses well above healthy control levels (P<0.05 in serum and P<0.01 in erythrocytes, compared to the control group). In sharp contrast, H₂O₂-scavenging and thioldependent recycling enzymes were severely compromised by the hypercaloric diet. Serum and erythrocytic catalase activities declined markedly in the untreated HFHS group, indicating a critical depletion of systemic peroxide detoxification capacity (P<0.05 in serum and P<0.01 in erythrocytes), while glutathione reductase (GR) activity declined significantly, compromising GSH recycling machinery (P<0.05 in serum and P<0.01 in erythrocytes). Mussel byssus administration rescued this network, robustly restoring both catalase and GR activities across circulating fractions though these restorations did not reach statistical significance in either serum or erythrocytes.

Table 7.

Impacts of Mytilus galloprovincialis byssus extract on serum antioxidant markers

Parameter	Control	HFHS	HFHS-by
SOD (U/mL)	18.50±2.80	22.10±3.40ns	32.40±5.09*ns
Catalase (U/mL)	15.19±2.11	7.80±1.32*	11.90±1.72ns
Glutathione reductase (nmol/min/mL)	45.01±6.01	24.00±4.09*	35.92±5.10ns

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

*P<0.05 vs. control group. ns, not significant.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus; SOD, superoxide dismutase.

Table 8.

Protective effects of Mytilus galloprovincialis byssus against oxidative stress in erythrocytes

Parameter	Control	HFHS	HFHS-by
SOD (U/mg Hb)	128.00±15.01	171.99±21.14ns	244.99±28.07**ns
Catalase (µmol H2O2/min/mg Hb)	72.00±8.03	35.00±6.12**	52.01±7.09ns
Glutathione reductase (nmol/min/mg Hb)	16.50±1.81	8.90±1.20**	13.20±1.50ns

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

**P<0.01 vs. control group. ns, not significant.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus; SOD, superoxide dismutase.

Although these parameters remained moderately below healthy control baselines, their reactivation is consistent with the hypothesis that the marine supplement may help stabilize circulating thiol-dependent defenses and protect erythrocyte membranes from oxidative structural decay, although membrane integrity was not directly assessed.

At the target-tissue level, the hypercaloric regimen severely impaired hepatic antioxidant capacities, reflecting localized oxidative depletion (Table 9). Unlike the circulatory compartments, hepatic SOD activity was modestly reduced in HFHS rats, although this decrease failed to reach statistical significance, suggesting a subclinical attenuation of the primary superoxide-related defense within the liver parenchyma. This effect was accompanied by a severe depletion of hepatic catalase activity (P<0.001), signaling a critical failure of peroxisomal H₂O₂ detoxification, and a marked decline in hepatic GR activity (P<0.001). Remarkably, byssus supplementation exerted a powerful hepatoprotective influence, reversing tissue-level impairment. In the HFHS-by group, hepatic SOD numerically exceeded that of control animals, although this elevation did not attain statistical significance relative to the control group; nevertheless, a significant upregulation was evident compared with the HFHS group (P<0.05), compensatory upregulation that ultimately surpassed baseline control values P<0.05 vs. HFHS, though not significant vs. control group, with remarkably restored hepatic catalase (P<0.01) and GR (P<0.001) activities. This targeted reinforcement of defensive measures underscores the capacity of the byssus matrix to mitigate local lipotoxicity and suppress diet-induced hepatic tissue injury.

Table 9.

Effects of Mytilus galloprovincialis byssus on hepatic antioxidant status

Parameter	Control	HFHS	HFHS-by
SOD (U/mg protein)	2.11±0.57	1.61±0.29ns	4.61±1.09ns#
Catalase (µmol H2O2 decomposed/min/mg protein)	56.68±2.34	25.03±1.39***	34.20±0.95***##
Glutathione reductase (µmol NADPH oxidized/min/mg protein)	34.37±0.30	18.12±0.35***	22.05±0.23***##

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01, and ^###P<0.001 vs. HFHS group. ns, not significant.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus; SOD, superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate.

The evaluation of serum PON1 arylesterase activity, an HDL-associated enzyme critical for preventing lipoprotein oxidation, further clarified the systemic impacts of byssus treatment (Table 10). In the untreated HFHS group, PON1 was markedly suppressed (P<0.001 vs. control group), reflecting either a severe oxidative inactivation of the enzyme or structural destabilization of the host HDL particles under chronic metabolic stress. Addition of mussel byssus markedly counteracted this decline, driving a substantial recovery of PON1 arylesterase activity toward healthy baseline values (P<0.001 vs. HFHS group); nonetheless, a residual and statistically significant deficit persisted relative to healthy control animals (P<0.01 vs. control group), indicating that, although byssus supplementation conferred substantial enzymatic protection, complete normalization of circulating PON1 activity was not achieved under the present dietary and dosing conditions. This stabilization aligns with current paradigms, suggesting that antioxidant-rich interventions preserve the structural and functional integrity of HDL particles, thereby maintaining their intrinsic antioxidant capacity and lowering overall atherogenic risk in metabolic syndrome models (Bhatti et al., 2022; Caturano et al., 2023).

Table 10.

Variations among experimental groups in serum paraoxonase-1 activity

Parameter	Control	HFHS	HFHS-by
Paraoxonase-1 (U/min/mL)	65.72±2.29	43.09±1.46***	56.11±1.31**###

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

**P<0.01 and ***P<0.001 vs. control group; ^###P<0.001 vs. HFHS group.

Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus.

The comprehensive antioxidant efficacy of M. galloprovincialis byssus is intimately linked to its unique struc tural composition, which is exceptionally rich in precollagens (PreCol-D, PreCol-NG, and PreCol-P), Gly-Pro-X triple-helical structures, and L-DOPA-containing residues. Gastrointestinal processing of this natural macromolecular matrix likely releases short, highly bioavailable peptides and catechol-bearing motifs, mirroring functionally relevant fractions identified in other marine organism-derived collagen sources (De Luca et al., 2016; Rahman, 2019; Geahchan et al., 2022). Peptides derived from the Gly-Pro-X-rich domains enriched with glycine, proline, Hyp, lysine, and arginine generally possess robust intrinsic radical-scavenging properties and are capable of chelating transition metal catalysts (Fe²⁺ and Cu²⁺), potentially halting Fenton-type propagation reactions. These combined biochemical traits may plausibly explain the containment of lipid peroxidation observed in byssus-treated animals, consistent with oral marine collagen hydrolysate interventions in dyslipidemic models (Suleria et al., 2015; De Luca et al., 2016; Geahchan et al., 2022).

Beyond direct radical scavenging, the increases in SOD, catalase, and GR activities observed in byssus-fed animals suggest a potential enhancement of intrinsic antioxidant defense systems. Consistent with mechanisms reported in the literature, marine-derived bioactive peptides in byssus could modulate redox-sensitive pathways, including the Nrf2/ARE axis, which regulates the transcription-level expression of multiple antioxidant enzymes. However, it must be emphasized that this mechanistic interpretation remains hypothetical in the context of this study, as no direct molecular evidence, such as Nrf2 expression or protein quantification, was obtained. Future investigations incorporating such analyses would be necessary to confirm whether the Nrf2/ARE pathway is involved in the antioxidant effects observed post-byssus supplementation (Suleria et al., 2015; Šimat, 2021; Geahchan et al., 2022). Furthermore, based on its known chemical properties, the unique catechol architecture of embedded DOPA residues is expected to provide sequential electron-donation capacities, potentially stabilizing free radicals and reinforcing systemic redox homeostasis (Cadar et al., 2024; Chen et al., 2025).

Although the restorations remain partial, a consistent trend toward the normalization of multiple antioxidant enzymes and PON1 activity across tissues underscores the potential of byssus as a complementary antioxidant strategy in diet-induced dyslipidemia and hyperglycemia.

Effect of mussel byssus on inflammatory cytokines

Inflammatory pathways play a central role in the pathogenesis of complications associated with hyperglycemia and hyperlipidemia, in close interplay with oxidative stress. Among these pathways, TNF-α and IL-6 are key pro-inflammatory cytokines involved in the amplification of systemic inflammation, insulin resistance, and tissue injury during metabolic syndrome, whereas CRP serves as a sensitive marker of the hepatic acute-phase inflammatory response (Ridker et al., 1997; Sifuentes-Franco et al., 2018).

Chronic HFHS dietary exposure induced a robust lowgrade inflammatory state, as evidenced by substantial elevations in serum TNF-α (P<0.001), IL-6 (P<0.05), and CRP (P<0.01) levels compared with control animals (Table 11). Such a pronounced pro-inflammatory profile reflects the well-established capacity of chronic highcalorie diets to promote systemic inflammation through lipotoxicity, postprandial hyperglycemia, and oxidative stress-driven immune activation. Dietary supplementation with Mediterranean mussel byssus significantly attenuated this inflammatory response, as evidenced by a marked reduction in serum TNF-α (P<0.01 vs. HFHS group, however, serum TNF-α levels remained significantly elevated relative to control animals (P<0.01 vs. control group), indicating partial rather than complete resolution of the pro-inflammatory phenotype. The decreases in IL-6 and CRP did not reach statistical significance relative to either the HFHS or the control group (ns for both comparisons) (Table 11).

Table 11.

Effects of Mytilus galloprovincialis byssus on TNF-α, IL-6, and CRP levels in HFHS diet-induced hyperlipidemic and hyperglycemic rats

Parameter	Control	HFHS	HFHS-by
TNF-α (pg/mL)	15.17±1.89	32.84±1.14***	25.01±1.67**##
IL-6 (pg/mL)	10.64±3.24	25.20±4.80*	18.71±1.23ns
CRP (mg/L)	0.70±1.32	4.80±1.27**	2.91±0.62ns

Values are presented as mean±standard error of the mean (n=8 per group).

Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test.

*P<0.05, **P<0.01, and ***P<0.001 vs. control group; ^##P<0.01 vs. HFHS group. ns, not significant.

TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; CRP, C-reactive protein; Control, control group fed a standard diet; HFHS, high-fat, high-sucrose diet-fed group; HFHS-by, high-fat, high-sucrose diet-fed group supplemented with 12% dried and finely ground mussel byssus.

Notably, however, CRP was quantified utilizing a semiquantitative latex agglutination method, and these values must therefore be interpreted as indicative of inflammatory trends rather than precise absolute concentrations. Although all inflammatory markers remained above basal physiological levels, the overall pattern demonstrates a prominent immunomodulatory potential of byssus in restraining the systemic inflammatory burden induced by metabolic stress.

The anti-inflammatory activity of byssus appears to be closely linked to its oxidative stress-mitigating capacity, as ROS-mediated signaling plays a pivotal role in activating inflammatory pathways. The attenuation of oxidative stress in byssus-treated animals likely suppresses the expression of pro-inflammatory cytokines, consistent with mechanisms reported in the literature, whereby marinederived bioactive peptides have been associated with a modulation of redox-sensitive inflammatory signaling. However, it has to be emphasized that the precise molecular mechanisms underlying these anti-inflammatory impacts, including the potential involvement of NF-κB and mitogen-activated protein kinase signaling pathways, remain hypothetical in the present context, as no direct molecular evidence was available. Future investigations incorporating gene expression and protein analyses are necessary to confirm these proposed pathways (Šimat, 2021; Geahchan et al., 2022; Gunasekara et al., 2025).

Overall, M. galloprovincialis byssus exerts a robust, albeit partial, anti-inflammatory effect likely mediated through the improvement of redox homeostasis. The functional interplay between antioxidant activity and inflammatory pathway modulation further underscores the relevance of byssus as a promising source of nutraceutical biomolecules for the adjunctive management of chronic metabolic disorders associated with oxidative stress and inflammation.

The present study has several limitations that must be acknowledged. The lack of histological analyses, including hematoxylin-eosin and Oil Red O staining, precludes the direct morphological assessment of diet-induced tissue injury and its attenuation by byssus supplementation. Furthermore, as this study relied exclusively on biochemical measurements and enzyme assays, the proposed mechanistic interpretations remain hypothetical and inferential. Future investigations should incorporate histopathological evaluations alongside gene expression profiling and protein quantification to fully validate the methodological hypotheses proposed herein and to establish a more comprehensive understanding of the therapeutic potential of mussel byssus in metabolic syndrome.

In conclusion, the findings demonstrate that M. galloprovincialis byssus exhibits significant therapeutic potential against metabolic alterations induced by an HFHS diet. Byssus markedly improved metabolic balance, attenuated oxidative stress, and limited inflammatory responses, as evidenced by complementary biochemical analyses. These benefits are likely attributable to the rich bioactive profile of byssus, including collagen peptides and antioxidant-activity residues. Nevertheless, further molecular and histopathological investigations are warranted to fully validate the mechanistic hypotheses proposed herein and to substantiate the potential of byssus as a marine-derived functional food ingredient for the management of metabolic syndrome.