Structural and Functıonal Characterızatıon of an Alcalase-Derıved Proteın Hydrolysate from Rapana Venosa for Functıonal Food Applıcatıons With Elemental Safety Assessment

Structural and Functıonal Characterızatıon of an Alcalase-Derıved Proteın Hydrolysate from Rapana Venosa for Functıonal Food Applıcatıons With Elemental Safety Assessment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Structural and Functıonal Characterızatıon of an Alcalase-Derıved Proteın Hydrolysate from Rapana Venosa for Functıonal Food Applıcatıons With Elemental Safety Assessment Neslihan AKYURT, Koray KORKMAZ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8912550/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Rapana venosa is a benthic marine gastropod with high protein content; however, its use as a food-derived protein source is limited by concerns regarding potentially toxic elements. In this study, a protein hydrolysate was produced from R. venosa muscle using Alcalase and characterized in terms of structural, functional, and elemental properties. The hydrolysate showed a high degree of hydrolysis (55.5%), supported by amino acid profiling and SDS-PAGE analysis indicating the predominance of low-molecular-weight peptides. The amino acid composition revealed a substantial proportion of essential and functional amino acids, with glutamic and aspartic acids as dominant components. The hydrolysate exhibited notable antioxidant activity, achieving 55.17% DPPH radical scavenging at 1 mg/mL. No significant reducing capacity was observed in the CUPRAC assay, suggesting that antioxidant activity is mainly associated with radical scavenging rather than metal ion reduction. No antimicrobial activity was detected against Staphylococcus aureus or Escherichia coli, consistent with the association of antimicrobial defense in mollusks with hemolymph-derived components rather than muscle tissue. Elemental analysis indicated the absence of chromium and cadmium, while lead and mercury were present at low levels. Nutritionally relevant minerals, including magnesium, iron, zinc, and copper, were retained in the protein-rich fraction. These findings demonstrate that enzymatic hydrolysis of R. venosa muscle yields a structurally and functionally favorable protein ingredient with a controlled elemental profile. Rapana venosa Protein hydrolysate Enzymatic hydrolysis Alcalase Antioxidant activity Heavy metals Figures Figure 1 Introduction Marine-derived proteins have attracted increasing attention in the fields of food and nutrition in recent years owing to their high biological value, balanced amino acid profiles, and diverse functional properties. The growing global demand for protein, together with the need for sustainable and alternative raw materials, has necessitated the evaluation of marine organisms beyond conventional fish species. In this context, protein hydrolysates obtained through enzymatic hydrolysis have emerged as promising ingredients, as their enhanced solubility, bioavailability, and functional potential render them valuable not only from a nutritional standpoint but also for technological applications (Cunha and Pintado 2022). Protein hydrolysates are produced through controlled enzymatic hydrolysis, during which protein chains are cleaved into shorter peptides and free amino acids, resulting in substantial modifications in digestibility and functional behavior. Numerous studies have reported that low-molecular-weight peptides are particularly advantageous with respect to functional properties, especially antioxidant activity. However, the magnitude and nature of these functional attributes are strongly influenced by the type of enzyme employed, hydrolysis conditions, and the structural characteristics of the raw material (García-Moreno et al. 2014). Consequently, the assessment of each novel marine resource in the form of protein hydrolysates offers the potential to yield distinct structural and functional outcomes. Rapana venosa has attracted attention as an alternative marine protein source due to its benthic lifestyle, high protein content, and wide geographical distribution. Nevertheless, its close interaction with sediment has placed this species in a controversial position with regard to the accumulation of heavy metals and potentially toxic elements (PTEs). Numerous studies have reported variable levels of Cd, Pb, As, and Hg in different tissues of R. venosa, with Cd often detected at comparatively higher concentrations than other metals in certain samples (Gedik, 2018; Önel et al. 2025; Ryabushko et al. 2022; Zhelyazkov et al. 2018). In contrast, Hg and Pb have frequently been reported as either undetectable or present at concentrations below the maximum permissible limits defined by national and international regulations (Altuğ and Güler 2002; Levent and Oztekin 2016). Previous studies have further demonstrated that metal accumulation in R. venosa varies markedly depending on tissue type. While metabolically active tissues such as the operculum and hepatopancreas generally exhibit higher metal concentrations, the edible muscle tissue is often reported to contain lower levels (Alkan and Alkan 2023). However, several investigations have indicated that Cd and Pb concentrations in muscle tissue may exceed regulatory limits under certain conditions, raising concerns regarding potential public health risks associated with long-term consumption (Gedik 2018; Önel et al. 2025; Ryabushko et al. 2022). Risk assessment approaches based on target hazard quotient (THQ), total THQ, and metal pollution index (MPI) have generally suggested low acute risk, while emphasizing that chronic exposure to Cd and As warrants careful consideration (Alkan and Alkan 2023, 2021; Bat et al. 2023; Levent and Oztekin 2016). Despite this extensive body of literature, the majority of existing studies have focused on evaluating heavy metal content in raw R. venosa tissues, whereas the influence of protein processing on the partitioning of metals into protein-rich fractions has received limited attention. Moreover, investigations into the functional properties of R. venosa-derived proteins have predominantly centered on hemolymph-derived antimicrobial compounds (Dolashka et al. 2011; Kirilova et al. 2024; Krumova et al. 2021), hemocyanin-derived components (Dolashka et al. 2016; Dolashka-Angelova et al. 2008), and gelatin-like products (Gaspar-Pintiliescu et al. 2019). In contrast, studies examining the edible muscle tissue—which represents a protein-rich and widely consumed fraction—as a functional ingredient remain scarce. This discrepancy underscores a significant knowledge gap regarding the functionalization of R. venosa muscle proteins. Accordingly, the evaluation of protein hydrolysates not only as functional components but also as products with a more controlled elemental profile represents an important and underexplored research area. In particular, whether a marine species commonly associated with environmental risk concerns can be transformed into a functional protein source through enzymatic hydrolysis, while simultaneously addressing safety-related considerations, remains a critical question. The present study aims to comprehensively investigate the structural, functional, and safety-related characteristics of a protein hydrolysate obtained from Rapana venosa muscle using Alcalase. To this end, the degree of hydrolysis, amino acid composition, molecular weight distribution, color properties, antioxidant and antimicrobial activities, as well as elemental and heavy metal contents were evaluated. This work seeks to elucidate the potential of a protein-rich yet safety-challenged marine resource to be valorized as a functional protein component for food applications through enzymatic hydrolysis, while explicitly considering its associated safety profile. Materials and Methods MATERIAL Rapana venosa muscle used as the research material was obtained from a marine snail processing and packaging facility operating in the Fatsa district of Ordu Province, Türkiye. The samples were divided into approximately 1 kg portions, placed in insulated polystyrene boxes containing ice, and transported to the Processing Technology Laboratory of the Department of Fisheries Technology Engineering, Faculty of Marine Sciences, Ordu University, under cold-chain conditions. Upon arrival, the samples were stored in frozen form at −20 °C until use in protein hydrolysate production. Hydrolysate Production Frozen Rapana venosa muscle samples were thawed at room temperature prior to analysis, then minced and homogenized using a meat grinder (Empero E.M.P.12.01.P). To eliminate the potential contribution of endogenous enzymes to the hydrolysis process, the homogenized samples were heat-treated in a water bath at 90 °C for 20 min to ensure endogenous enzyme inactivation. After thermal treatment, the samples were cooled and diluted with distilled water at a ratio of 1:1 (w/v), followed by further homogenization to obtain a suitable matrix for enzymatic hydrolysis. Protein hydrolysate production was carried out based on the enzymatic hydrolysis method optimized by Korkmaz (2018). A commercial alkaline protease, Alcalase, was used as the hydrolyzing enzyme. The optimum hydrolysis temperature, pH range, and enzyme inactivation conditions were applied in accordance with the technical specifications provided by the manufacturer. Accordingly, enzymatic hydrolysis was performed at 50 °C, and enzyme inactivation was achieved by heating at 85 °C for 10 min. In this study, hydrolysis conditions were applied based on previously optimized parameters targeting protein hydrolysis degree and process yield, rather than optimization toward a specific biological activity. According to the method described by Korkmaz (2018), the homogenized samples were hydrolyzed at 50 °C using an enzyme concentration of 1% (w/w) and a hydrolysis time of 1 h. Following hydrolysis, enzymatic activity was terminated by placing the reaction mixtures in a water bath at 85 °C for 10 min, after which the samples were cooled at room temperature for 15 min. The cooled hydrolysate solutions were then centrifuged at 4000 rpm for 20 min to achieve phase separation. No distinct lipid phase was observed after centrifugation; instead, three fractions were obtained: an upper fraction containing minor protein–lipid components, a middle aqueous fraction rich in protein, and a bottom fraction consisting of insoluble materials. The protein-rich middle fraction was used for the characterization and subsequent analyses of the Rapana venosa protein hydrolysate. Proximate Composition Analysis The proximate composition of the protein hydrolysate was evaluated by determining crude protein, crude lipid, moisture, and ash contents. Crude protein content was determined based on total nitrogen using the Kjeldahl method (N × 6.25) according to AOAC (1998). Moisture content was analyzed following AOAC (1990), and ash content was determined according to AOAC method 935.47 (1998). Total lipid content was determined using the method of Bligh and Dyer (1959). Briefly, 15 g of protein hydrolysate sample was homogenized, and 120 mL of a methanol/chloroform mixture (1:2, v/v) was added. The mixture was blended using a Waring blender, followed by the addition of 20 mL of 0.4% (w/v) CaCl₂ solution. The resulting suspension was then filtered through filter paper (Schleicher & Schuell, 595 1/2, 185 mm). The filtrate was transferred into pre-weighed round-bottom flasks, which were sealed to prevent solvent evaporation and stored in the dark overnight. After this period, the upper phase consisting of methanol and water was removed using a separatory funnel. The remaining chloroform phase was evaporated at 60 °C in a water bath using a rotary evaporator (Heidolph Basis Hei-Vap ML). The residual lipid fraction was further dried in an oven at 60 °C for 1 h to ensure complete removal of chloroform residues (Termal G11540SD). The flasks were then cooled to room temperature in a desiccator and weighed using an analytical balance with a sensitivity of 0.1 mg (Precisa XB 220A). Total lipid content was calculated using the following equation: Lipid content (%) = [(Weight of flask + lipid) − Weight of empty flask] × 100 / Sample weight (g) Degree of Hydrolysis The degree of hydrolysis (DH, %) of the protein hydrolysate was determined using the trichloroacetic acid (TCA) solubility method described by Hoyle and Merritt (1994). This method is based on the solubility of low-molecular-weight peptides and free amino acids generated during enzymatic hydrolysis and is widely used for assessing the extent of protein hydrolysis in protein hydrolysates. Following the completion of enzymatic hydrolysis, the hydrolysate was mixed with 20% (w/v) TCA solution at a ratio of 1:1 (v/v). The mixture was then centrifuged at 15,000 × g for 20 min at 4 °C. After centrifugation, the clear supernatant containing proteins and peptides soluble in 10% TCA was collected, and all analyses were performed in triplicate. The degree of hydrolysis was calculated based on the ratio between the amount of protein soluble in 10% TCA and the total protein content of the hydrolysate. Total protein content was determined using the Kjeldahl method. The degree of hydrolysis was expressed according to the following equation: DH (%) = (N₀ / Nᵀ) × 100 where N₀ represents the amount of protein soluble in 10% TCA, and Nᵀ represents the total protein content of the hydrolysate. In this study, the degree of hydrolysis was evaluated to assess the effectiveness of the applied hydrolysis conditions and the extent of protein conversion into low-molecular-weight fractions; no optimization targeting specific biological activity was conducted. Electrophoresis The molecular weight distribution of the protein hydrolysate was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The analyses were performed according to the discontinuous gel electrophoresis system described by Laemmli (1970). For this purpose, protein hydrolysate samples were diluted to an appropriate concentration and mixed with sample buffer containing SDS, glycerol, Tris–HCl, and bromophenol blue. The samples were heated at 95 °C for 5 min to ensure complete protein denaturation. After cooling, the samples were loaded onto polyacrylamide gels consisting of a 4% stacking gel and a 12% resolving gel. Electrophoresis was carried out under constant voltage conditions to allow separation of protein fractions based on their molecular weights. Upon completion of electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 to visualize protein bands and subsequently destained using an appropriate solvent to remove background staining. The molecular weights of the protein bands were estimated by comparison with a concurrently run protein molecular weight marker. SDS-PAGE analysis was employed to visually assess the conversion of proteins into lower-molecular-weight peptide fractions as a result of enzymatic hydrolysis. Color The color properties of the film samples obtained from the protein hydrolysate were determined using the CIE Lab* color space, where L* represents lightness, a* represents the red–green axis, and b* represents the yellow–blue axis. Color measurements were carried out using a Konica Minolta CM-5 colorimeter (Osaka, Japan). Measurements were performed to evaluate the homogeneity of the film surface and to assess the effect of the hydrolysis process on the visual characteristics of the samples. For each film sample, color values were recorded at multiple points on the surface, and the mean values were used for further analysis. Heavy Metal Determination The determination of heavy metals and mineral elements in the Rapana venosa protein hydrolysate was carried out using inductively coupled plasma mass spectrometry (ICP-MS). The analyses were performed through an accredited analytical laboratory specializing in elemental analysis. Measurements were conducted to determine the elemental composition and potential heavy metal content of the protein hydrolysate. Analytical results were calculated on a dry weight basis and expressed as mg/kg. The obtained data were used to evaluate the effect of the enzymatic hydrolysis process on the transfer of heavy metals potentially present in Rapana venosa muscle tissue into the hydrolysate fraction. Total and Free Amino Acid Analysis The total amino acid composition of the Rapana venosa protein hydrolysate was determined based on the methods reported by Lee and Hwang (2017) and Chan and Matanjun (2017), with appropriate modifications to adapt the procedure to the protein hydrolysate matrix. Amino acid profiling was performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), which provides high sensitivity and selectivity (Thermo Fisher Scientific Inc., Waltham, MA, USA). For this purpose, 0.2 g of the protein hydrolysate sample was homogenized and weighed into a solution containing 10 mL of 6 N HCl. To prevent the loss of volatile components during hydrolysis, the samples were tightly sealed in test tubes, vortexed for 5 min, and hydrolyzed in an oven at 110 °C for 24 h to ensure complete cleavage of proteins into their constituent amino acids. After hydrolysis, the samples were cooled to room temperature and centrifuged at 4000 rpm for 15 min at 4 °C. The clear supernatant obtained after centrifugation was filtered through a 0.45 µm PTFE membrane filter to remove particulate matter and subsequently injected into the LC-MS/MS system for analysis. The resulting amino acid profile was used to evaluate the nutritional quality of the protein hydrolysate and to assess the free amino acids released as a result of enzymatic hydrolysis. Antioxidant Activity The antioxidant activity of the protein hydrolysate was evaluated using two complementary methods based on different antioxidant mechanisms: CUPRAC (Cupric Ion Reducing Antioxidant Capacity) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assays. Prior to analysis, protein hydrolysate samples were dissolved in distilled water at a concentration of 1 mg/mL, and all measurements were performed in triplicate. For the CUPRAC assay, the reagent was freshly prepared by mixing equal volumes of 10 mM CuCl₂, 7.5 mM neocuproine (prepared in ethanol), and 1 M ammonium acetate buffer (pH 7.0). The hydrolysate solution was mixed with the CUPRAC reagent, and the reaction mixture was incubated at room temperature for 30 min in the dark. After incubation, absorbance values were measured at 450 nm using a UV–Vis spectrophotometer. Antioxidant capacity was calculated based on a Trolox standard curve and expressed as µmol Trolox equivalents (TE)/g hydrolysate. DPPH radical scavenging activity was determined using a 0.1 mM DPPH solution prepared in methanol. The hydrolysate solution was mixed with the DPPH solution to a final volume of 1.0 mL and vortexed thoroughly. The mixture was incubated at room temperature for 30 min under dark conditions. The decrease in absorbance of the purple DPPH radical, resulting from its interaction with antioxidant components in the protein hydrolysate, was measured at 517 nm using a UV–Vis spectrophotometer. DPPH radical scavenging activity was calculated based on the reduction in absorbance relative to the control, and the results were expressed as µmol TE/g hydrolysate using a Trolox standard curve. The antimicrobial activity of the protein hydrolysates was evaluated using the agar well diffusion method against two reference bacterial strains: one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Bacterial cultures were activated on Mueller–Hinton Agar (MHA) and adjusted to the 0.5 McFarland standard (approximately 1 × 10⁸ CFU/mL). The prepared bacterial suspensions were uniformly spread onto the surface of MHA plates using sterile cotton swabs. To prevent microbial contamination, protein hydrolysate solutions were sterilized by filtration through 0.22 µm pore-size membrane filters. The final concentrations used in the assay were prepared in the range of 25–50 mg/mL. Wells with a diameter of 6 mm were aseptically punched into the agar surface, and 50–100 µL of the hydrolysate solutions were added to each well. To allow diffusion of the hydrolysates into the agar medium, the plates were kept at room temperature for 30 min prior to incubation, followed by incubation at 37 °C for 24 h. After incubation, clear zones indicating bacterial growth inhibition were measured using a digital caliper, and antimicrobial activity was recorded as inhibition zone diameter (mm). Ampicillin (10 µg) was used as a positive control, while the solvent used for dissolving the hydrolysates (sterile water or 50% ethanol) served as the negative control. The antimicrobial assay was conducted to delineate the potential biological activity boundaries of the protein hydrolysate. RESULTS AND DISCUSSION Proxymate Composition The hydrolysate exhibited a protein-rich composition with a high protein content (75.5%) and a low lipid level (3.4%), indicating a matrix enriched in protein while being limited in lipid fraction. This composition suggests that the applied enzymatic hydrolysis and subsequent fractionation steps were effective in enriching the protein fraction. The relatively high ash content (12.6%) can be attributed to the characteristic mineral composition of marine-derived raw materials. Similarly, the literature reports that ash content in various marine protein hydrolysates may vary over a wide range, depending on the species, tissue type, and processing conditions applied (Henriques et al. 2021). Based on these proximate composition findings, the predominance of the protein fraction indicates that the matrix provides a quantitatively substantial protein source from which short-chain peptides and free amino acids associated with functional activity may be released during enzymatic hydrolysis. The extent to which this potential is realized is elucidated through the evaluation of post-hydrolysis structural transformations. Degree of Hydrolysis (%HD) In the present study, the degree of hydrolysis of the protein hydrolysate obtained from Rapana venosa muscle was determined to be 55.5%. This value indicates that peptide bonds within the protein chains were extensively cleaved during enzymatic hydrolysis, reflecting a high level of enzymatic breakdown. The degree of hydrolysis is considered a critical parameter in the production of protein hydrolysates, as it represents a fundamental indicator of the extent of peptide bond cleavage from the parent protein (Benjakul and Morrissey 1997). The percentage degree of hydrolysis (%DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate and is widely accepted as one of the most important measures of hydrolysis efficiency. It has been reported in the literature that an increase in the degree of hydrolysis is associated with enhanced protein recovery, the formation of lower molecular weight peptides due to protein chain fragmentation, and a consequent improvement in water solubility, ultimately leading to enhanced functional properties of the hydrolysate (Shahidi et al. 1995). Alcalase has been widely reported to achieve higher degrees of hydrolysis compared to other commercial proteases. Ovissipour et al. (2012) investigated the hydrolysis of Persian sturgeon proteins using five different enzymes (Alcalase, Protamex, Neutrase, Flavourzyme, and Trypsin) and reported that the highest degree of hydrolysis was obtained with Alcalase. Similarly, Yoon et al. (2015) hydrolyzed liver extracts from Oncorhynchus keta and Oncorhynchus gorbuscha using various commercial enzymes and demonstrated that the highest soluble protein recovery was achieved with Alcalase at 50 °C and pH 7.0. In a comparative study conducted on rice bran protein concentrate and soy protein, Ahmadifard et al. (2016) also reported that Alcalase exhibited a higher hydrolytic capacity than other enzymes. Overall, the literature indicates that Alcalase typically produces degrees of hydrolysis ranging from approximately 16% to 67% in proteins of aquatic origin (Edison et al. 2020; Mohanty et al. 2021; Pires et al. 2024; Rajabzadeh et al. 2018; Sierra-Lopera et all. 2021). This behavior is attributed to the enzyme’s high catalytic efficiency and broad substrate specificity. The 55.5% degree of hydrolysis determined for Rapana venosa in the present study falls within the upper range of reported values, indicating efficient protein cleavage and suggesting that an appropriate hydrolysis level was achieved for the generation of biologically valuable low-molecular-weight peptide fractions. Total and Free Amino acids The amino acid composition of the protein hydrolysate obtained from Rapana venosa muscle was evaluated by separately determining total and free amino acid fractions. This approach enabled the assessment of both the overall nutritional profile and the extent to which enzymatic hydrolysis promoted the release of amino acids into the free form. The results are summarized in Table 1. Amino Acid Total AA (mg/g) Free AA (mg/g) Alanine 14.80 5.38 Glycine 7.06 2.54 Threonine* 7.15 1.85 Lysine* 16.17 6.01 Arginine 17.56 4.90 Histidine* 2.34 0.73 Aspartic acid 21.61 7.39 Cystine 0.03 0.01 Glutamic acid 48.75 15.08 Serine 6.84 2.51 Proline 3.70 0.95 Hydroxyproline 1.44 0.39 Valine* 17.51 5.24 Methionine* 0.94 0.30 Tyrosine 0.67 0.24 Leucine* 13.65 4.75 Isoleucine* 3.45 1.27 Asparagine 0.02 0.01 Phenylalanine* 0.63 0.26 * Essential amino acids (EAA) Table 1. Amino acid composition of the protein hydrolysate obtained from Rapana venosa muscle, expressed as total and free amino acid fractions (mg/g). The predominance of glutamic acid and aspartic acid in both total and free amino acid distributions demonstrates that the hydrolysate is rich in amino acids bearing acidic side chains. The presence of these amino acids at high levels in the free fraction suggests that the post-hydrolysis structure is highly exposed to radical interactions. It has been reported that acidic amino acids make a strong contribution to free radical scavenging activity, while exhibiting limited involvement in metal-reduction–based antioxidant mechanisms (Udenigwe and Aluko 2011). With respect to basic amino acids, lysine and arginine were detected at considerable levels in both the total and free fractions. Owing to the charged groups present in their side chains, these amino acids are capable of directly interacting with free radicals and forming complexes with metal ions (Aissaoui et al. 2017; Hu et al. 2020). The notable presence of lysine in the free amino acid fraction suggests that the hydrolysate acquires enhanced molecular reactivity and functional potential following enzymatic hydrolysis. Aromatic amino acids, namely phenylalanine and tyrosine, were identified in both total and free amino acid profiles, supporting their contribution to the radical scavenging behavior of the hydrolysate. Aromatic side chains are known to donate protons to electron-deficient radicals, rendering these amino acids key contributors to free radical quenching mechanisms (Rajapakse et al. 2005; Yang et al. 2019). Their occurrence in the free fraction further indicates that antioxidant activity is not merely related to total protein content, but is strongly influenced by molecular accessibility achieved through hydrolysis. Among aliphatic amino acids, leucine, valine, and alanine were found at elevated levels in the free fraction, a result that is consistent with the characteristic proteolytic specificity of Alcalase. It has been reported that Alcalase effectively cleaves peptide bonds adjacent to aliphatic and aromatic amino acids, generating hydrolysates with enhanced biological activities, including antioxidant properties (Paek et al. 2001; Qian et al. 2008). Accordingly, the amino acid distribution observed in the present study reflects a structured enzymatic transformation driven by enzyme–substrate interactions rather than a nonspecific degradation process. The relatively low levels of sulfur-containing amino acids, such as cysteine and methionine, suggest that the antioxidant behavior of the hydrolysate is predominantly governed by acidic, basic, and aromatic amino acids. Although the thiol group of cysteine is known to exhibit strong antioxidant activity (Qian et al. 2008), the present findings indicate that the antioxidant response of the hydrolysate does not rely primarily on sulfur-containing residues. It is well established that the antioxidant properties of peptides are closely related to their amino acid composition, molecular structure, and hydrophobicity (Alemán et al. 2011; Chen et al. 1998). When the high abundance of free amino acids is considered alongside the dominance of low-molecular-weight fractions, the antioxidant behavior of the Rapana venosa protein hydrolysate can be interpreted as a direct consequence of increased molecular accessibility and reactivity resulting from enzymatic hydrolysis. Collectively, these findings demonstrate that the antioxidant activity of the hydrolysate can be coherently explained within a composition-driven and structure–activity–consistent framework. When the total and free amino acid profiles of the Rapana venosa protein hydrolysate are evaluated together, it becomes evident that the observed antioxidant behavior is not a random outcome, but rather is closely associated with the amino acid composition and the structural transformations induced by enzymatic hydrolysis. In particular, the pronounced enrichment of specific amino acids in the free fraction indicates that hydrolysis effectively occurred along the protein backbone, rendering functional building blocks accessible within the matrix. SDS-Page SDS-PAGE analysis was applied to the protein hydrolysate in order to elucidate the presence of peptide fractions formed in accordance with the determined degree of hydrolysis and to assess their molecular weight distribution. Examination of the electrophoretic profile obtained from SDS-PAGE analysis revealed a pronounced reduction in the bands corresponding to 25 and 70 kDa, accompanied by an evident increase in band intensity within the low-molecular-weight fractions. In particular, the predominance of bands below 25 kDa indicates that the applied enzymatic hydrolysis process resulted in an advanced level of protein chain cleavage. In the literature, a decrease in high-molecular-weight bands and the predominance of low-molecular-weight bands in SDS-PAGE profiles of protein hydrolysates are widely accepted as key indicators of increased degree of hydrolysis. The appearance of intense banding within the 10–25 kDa range or below following enzymatic hydrolysis has been reported as a characteristic outcome of successful and advanced hydrolysis processes (Zaky et al. 2019). In this context, the predominance of low-molecular-weight bands observed in the Rapana venosa protein hydrolysate is structurally consistent with the determined degree of hydrolysis of 55.5%. Bui et al. (2021) reported that bioactive peptides generally consist of 3–20 amino acid residues and predominantly possess molecular weights below 50 kDa. Raghavan and Kristinsson (2008) demonstrated that tilapia protein hydrolysates with molecular weights in the range of 3.5–10 kDa exhibited stronger antioxidant activity. Silvestre et al. (2013) classified the relationship between degree of hydrolysis and peptide size into four levels, indicating that hydrolysates with degrees of hydrolysis between 20% and 50% predominantly contain peptides smaller than 3 kDa and that this segment represents a suitable range for the generation of bioactive peptides. This classification is directly consistent with the 55.5% degree of hydrolysis determined in the present study. Accordingly, evaluation of the RVPH indicates that high-molecular-weight protein structures were largely eliminated, while low-molecular-weight peptide fractions—widely regarded as more functional in both biological and technological contexts—became predominant. This structural transformation provides a coherent and robust framework for interpreting the functional behavior of the hydrolysate at the molecular level. Color Characteristics The color parameters of the Rapana venosa protein hydrolysate were determined as L* = 88.01, a* = −1.62, and b* = 15.16. These values indicate that the hydrolysate exhibits a light-colored appearance with high brightness and minimal chromatic contribution. The high L* value reflects a visually light and homogeneous product, while the negative a* value confirms the absence of red tonal components. The moderate b* value suggests a limited degree of yellow coloration. It has been reported that the color characteristics of protein hydrolysates vary widely depending on the raw material and the tissue source used. Thiansilakul et al. (2007) reported L* = 58.00, a* = 8.38, and b* = 28.3 for round scad protein hydrolysate, corresponding to a pronounced brownish–yellow color profile. Similarly, Kumari et al. (2023) determined L* = 57.1 ± 1.5, a* = 3.0 ± 0.1, and b* = 10.9 ± 0.5 for pink perch viscera protein hydrolysate powder, describing the product as dark with brownish–yellow tones. In the same study, head-derived protein hydrolysates exhibited higher L* and lower a* and b* values compared to viscera-derived samples. Consistently, Hassan et al. (2019) emphasized that protein hydrolysates obtained from visceral tissues generally display darker color profiles. The yellow–brown coloration commonly observed in protein hydrolysates has been largely attributed to intermediate or advanced Maillard reaction products. In this context, decreases in L* values accompanied by increases in a* and b* values are often associated with the presence of reactive proteins and free amino acids involved in non-enzymatic browning reactions (Kumari et al. 2023; Liu et al. 2004). In the present study, the simultaneous occurrence of a high L* value and a negative a* value indicates the absence of noticeable Maillard-related browning, suggesting that color-related chemical stability was preserved during processing. This finding distinguishes the Rapana venosa protein hydrolysate from many previously reported marine protein hydrolysates characterized by darker coloration. Furthermore, light-colored protein hydrolysates have been reported to exhibit higher visual acceptability compared to darker counterparts (Kumari et al. 2023). From this perspective, the neutral and light color profile of the Rapana venosa protein hydrolysate may be considered advantageous for its potential application in functional food formulations and protein-based industrial products where visual quality is an important factor. Antioxidant Activity In this study, the antioxidant capacity of the Rapana venosa protein hydrolysate was evaluated using two complementary assays based on different mechanisms of action: DPPH radical scavenging activity and CUPRAC (Cu²⁺ reducing capacity). It is well established that the antioxidant potential of protein hydrolysates and bioactive peptides is not restricted to a single mechanism, but may arise through multiple pathways, including free radical scavenging, hydrogen donation, and modulation of redox processes (Frankel and Meyer 2000). Therefore, the combined application of these two methods enables a mechanism-oriented evaluation of the antioxidant behavior of the hydrolysate. The DPPH assay demonstrated that the Rapana venosa protein hydrolysate exhibited a radical inhibition of 55.17% at a concentration of 1 mg/mL. This level of activity is higher than, or comparable to the upper range of, values reported for many marine protein hydrolysates, indicating a pronounced free radical scavenging capacity. Previous studies have shown that DPPH radical scavenging activities of protein hydrolysates derived from various fish and plant sources generally range between 15% and 45% (García-Moreno et al. 2014). Jamdar et al. (2010) and Wu et al. (2003) further reported that increases in the degree of hydrolysis enhance DPPH scavenging activity, primarily due to the generation of low-molecular-weight peptides and free amino acids. In this context, the relatively high inhibition observed in the present study suggests that the applied enzymatic hydrolysis conditions effectively promoted the release of antioxidant-active peptide fractions. When the DPPH results are evaluated together with amino acid composition and SDS-PAGE findings, a clear mechanistic explanation emerges. The hydrolysate was characterized by a predominance of low-molecular-weight peptide fractions and an amino acid profile enriched in electron-donating amino acids, including glutamic acid, aspartic acid, alanine, and glycine. These structural features are known to facilitate hydrogen donation and direct interactions with free radicals, thereby contributing to the stabilization of reactive species and the termination of radical chain reactions. Accordingly, the strong radical scavenging activity observed in the DPPH assay can be directly attributed to the peptide composition and molecular size distribution of the hydrolysate. In contrast, no notable reducing capacity was detected in the CUPRAC assay. This discrepancy between DPPH and CUPRAC results can be explained by the distinct antioxidant mechanisms underlying these methods. While the DPPH assay primarily reflects hydrogen donation and free radical scavenging ability, the CUPRAC method is based on the reduction of metal ions. Therefore, the absence of CUPRAC activity does not contradict the DPPH findings; rather, it indicates that the antioxidant behavior of the Rapana venosa protein hydrolysate is not associated with metal ion reduction. Instead, the results demonstrate a mechanism-specific antioxidant profile in which the antioxidant potential is predominantly governed by radical scavenging pathways linked to the structural characteristics of the peptide fractions. Antimicrobial Activity The antimicrobial activity of the protein hydrolysate obtained from Rapana venosa muscle was evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. The results demonstrated that the hydrolysate did not exhibit inhibitory activity against either of the tested bacterial strains. Previous studies have consistently reported that antimicrobial activity in mollusks is primarily associated with hemolymph-derived proteins and peptides rather than muscle tissue. In particular, hemocyanins and hemocyanin-derived peptides present in the hemolymph have been shown to display agglutinative and antibacterial properties against a range of pathogenic microorganisms (Dolashka et al. 2011; Krilova et al. 2024). In contrast, there is limited evidence in the literature supporting a comparable antimicrobial defense function for muscle tissue or edible flesh fractions. In this context, the absence of antimicrobial activity observed for the Rapana venosa muscle protein hydrolysate is fully consistent with the established understanding that antimicrobial defense mechanisms in mollusks are predominantly mediated by immune-related hemolymph components rather than structural tissues. Therefore, the lack of antimicrobial effect should not be regarded as a limitation of the hydrolysate, but rather as a biologically coherent outcome aligned with the tissue origin of the raw material and the current body of literature. Elemental Composition Table 2. Elemental composition of Rapana venosa muscle reported in the literature and the protein hydrolysate obtained in the present study (dry weight basis). Element Rapana venosa muscle (literature range, mg/kg, dry weight) Protein hydrolysate (this study, mg/kg) Cr 0.2 – 1.5 ND Cd 0.1 – 1.2 ND Pb 0.5 – 3.0 0.80 Hg 0.1 – 0.9 0.33 As 5 – 25 15.57 Ni 0.5 – 2.0 trace Co 0.1 – 0.8 trace Fe 150 – 450 172.30 Zn 40 – 120 40.29 Cu 10 – 60 27.73 Mg 800 – 3000 12,546.94 Data compiled from multiple literature sources (e.g., Alkan and Alkan, 2023; Altuğ and Güler 2002; Bayraklı et al. 2024; Gedik 2018; Levent and Öztekin, 2016; Mülayim and Balkıs 2015; Önel et al., 2025; Ryabushko et al. 2022; Zhelyazkov et al. 2018). The literature ranges represent the minimum and maximum concentrations reported for Rapana venosa muscle tissue across different studies and reflect variability associated with habitat characteristics, sampling location, and analytical methods. The heavy metal and mineral element contents of Rapana venosa meat and the protein hydrolysate obtained in this study are comparatively presented in Table 2. In the literature, heavy metal concentrations reported for Rapana venosa muscle tissue are known to vary widely, largely due to the benthic lifestyle of the species and its close interaction with sediment. In particular, potentially toxic elements such as Cd, Pb, As, and Hg have been reported at variable levels in raw tissues across different studies. In the present study, Cr and Cd were not detected (ND) in the protein hydrolysate, while Ni and Co were detected only at trace levels. In contrast, the concentrations of Pb (0.80 mg/kg), Hg (0.33 mg/kg), and As (15.57 mg/kg) were within, or close to the lower bounds of, the ranges previously reported for Rapana venosa meat in the literature. These findings suggest that the enzymatic hydrolysis process, together with subsequent centrifugation and fractionation steps, may limit the transfer of certain heavy metals into the protein-rich hydrolysate fraction. The absence of detectable Cd in the hydrolysate fraction is particularly noteworthy. Although Cd has been reported at relatively high levels in Rapana venosa tissues in some studies, its non-detectable level in the protein hydrolysate indicates that metal–protein interactions may exhibit tissue- and fraction-specific behavior. Similarly, the non-detection of Cr in the hydrolysate fraction suggests that a substantial portion of this element may remain associated with insoluble residues or fractions separated during the hydrolysis process. Comparable observations have been reported for protein hydrolysates derived from other marine species. Mangano et al. (2021) investigated the heavy metal content of anchovy protein hydrolysates and reported that the levels detected were below maximum limits. Similarly, la Fuente et al. (2023) examined protein hydrolysates obtained from mackerel and salmon and concluded that their heavy metal contents remained within acceptable safety ranges. In a broader evaluation, Wu et al. (2022) emphasized that fish protein hydrolysates are generally not considered a potential risk in terms of heavy metal content. These findings support the view that enzymatic hydrolysis may influence the distribution and bioavailability of metals within protein-rich fractions across different marine species. With respect to nutritionally essential elements, Fe (172.30 mg/kg), Zn (40.29 mg/kg), Cu (27.73 mg/kg), and especially Mg (12,546.94 mg/kg) were retained at appreciable levels in the hydrolysate fraction. This observation indicates that while the enzymatic hydrolysis process may restrict the migration of certain potentially toxic elements, it allows nutritionally valuable minerals to be preserved within the protein-rich fraction. Taken together, these results demonstrate that a marine resource such as Rapana venosa, which is often discussed in the literature in terms of heavy metal accumulation, can exhibit a more controlled and predictable elemental profile when processed into a protein hydrolysate via enzymatic hydrolysis. These findings highlight the importance of evaluating protein hydrolysates not only in terms of their functional properties but also with respect to their elemental safety profiles, and suggest that enzymatic hydrolysis may represent a promising processing strategy for the utilization of environmentally sensitive marine protein sources. Conclusion In this study, a protein hydrolysate was produced from Rapana venosa muscle tissue using Alcalase, and the resulting product was comprehensively evaluated in terms of proximate composition, degree of hydrolysis, amino acid profile, molecular weight distribution, color characteristics, antioxidant capacity, antimicrobial activity, and elemental/heavy metal content. The findings collectively demonstrate that the hydrolysate is not merely a protein-rich product, but rather exhibits a coherent and predictable profile in terms of composition–structure–function relationships. Proximate analyses revealed that the hydrolysate possessed a high protein content (75.5%) and a low lipid level (3.4%), indicating a distinctly protein-based matrix. The relatively high ash content (12.6%) is consistent with the characteristic mineral composition of marine-derived raw materials and aligns well with values reported for comparable marine protein hydrolysates in the literature. This fundamental composition indicates the presence of a quantitatively dominant protein source from which short-chain peptides and free amino acids associated with functional activity can be released during enzymatic hydrolysis. The degree of hydrolysis was determined to be 55.5%, demonstrating that the applied enzymatic treatment resulted in an advanced level of protein chain cleavage. This finding was structurally confirmed by SDS-PAGE analysis, which showed a pronounced reduction in high-molecular-weight bands and the predominance of bands below 25 kDa. According to the literature, this hydrolysis range represents a favorable segment for the generation of biologically active low-molecular-weight peptides. Accordingly, the molecular weight distribution of the obtained hydrolysate indicates that the structural prerequisites for functional activity were successfully established. Total and free amino acid analyses demonstrated that the hydrolysate contained essential amino acids contributing to nutritional quality, while also exhibiting a composition enriched in glutamic and aspartic acids, conferring sensory and technological advantages. The marked presence of the free amino acid fraction confirms that the hydrolysis process was not merely nominal, but effectively progressed at the molecular level, rendering functionally relevant chemical groups accessible within the matrix. This enables the functional behavior of the hydrolysate to be interpreted directly on the basis of experimentally determined compositional and structural data. Color measurements indicated that the hydrolysate exhibited a light and neutral appearance, characterized by a high L* value (88.01), a negative a* value (−1.62), and a moderate b* value (15.16). These results indicate the absence of pronounced Maillard-related browning during processing and suggest that the initial chemical stability of the product was preserved. The light color profile may therefore be considered advantageous for applications in functional food formulations and protein-based ingredient systems where visual acceptability is an important criterion. Functional evaluation revealed that the hydrolysate exhibited a DPPH radical inhibition of 55.17%, indicating a pronounced free radical scavenging capacity. In contrast, no significant reducing capacity was detected in the CUPRAC assay. This discrepancy demonstrates that the antioxidant behavior of the hydrolysate is governed primarily by radical scavenging and hydrogen-donating mechanisms rather than metal ion reduction. When considered together with the amino acid composition and the predominance of low-molecular-weight peptide fractions, the observed antioxidant activity can be explained within a composition-driven, mechanism-specific structure–activity framework. No antimicrobial activity was observed against Staphylococcus aureus or Escherichia coli. Given that antimicrobial defense mechanisms in mollusks are predominantly associated with hemolymph-derived components rather than muscle tissue, the absence of antimicrobial activity in the muscle-derived protein hydrolysate is biologically coherent and consistent with existing literature. One of the most notable outcomes of this study concerns the elemental and heavy metal profile of the hydrolysate. The non-detection of Cr and Cd, the presence of Pb and Hg at low levels, and the retention of essential minerals such as Mg, Fe, Zn, and Cu indicate that enzymatic hydrolysis and fractionation steps can limit the transfer of potentially toxic elements into the protein-rich fraction while preserving nutritionally valuable minerals. These findings suggest that a marine resource such as Rapana venosa, which is often discussed in the literature in relation to heavy metal accumulation, can be converted—under appropriate processing conditions—into a protein hydrolysate with a more controlled and predictable elemental profile. Overall, the results of this study demonstrate that the functional properties of Rapana venosa protein hydrolysate are not attributable to isolated analytical parameters, but rather emerge from the integrated effects of hydrolysis degree, molecular weight distribution, amino acid composition, and antioxidant behavior. Importantly, these functional outcomes are supported directly by experimentally measurable structural and compositional indicators, without reliance on theoretical assumptions or computational predictions. In conclusion, Rapana venosa protein hydrolysate represents a strong and multidimensional candidate for functional food formulations and protein-based ingredient applications, owing to its advanced hydrolysis characteristics, accessible and balanced amino acid composition, favorable color and stability profile, mechanism-specific antioxidant capacity, and improved heavy metal safety attributes. Future studies focusing on molecular-weight-based peptide fractionation and LC–MS/MS-driven peptidomic identification, as well as in vitro digestion and bioaccessibility assessments, would further strengthen the application potential of this hydrolysate. Declarations Author Contributions: K.K.: Methodology, Software (statistical analyses), Writing—original draft, Investigation. N.A: Conceptualization, Writing, Methodology. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data generated and analyzed during this study are included in this article. Conflicts of Interest: The authors declare no conflicts of interest. Clinical trial number: Not applicable, as this study did not involve human participants or clinical interventions. Acknowledgment: This study is derived from a part of the PhD thesis entitled “Investigation of the Effects of Chitosan-Based Edible Films Enriched with Rapana venosa Protein Hydrolysate on the Physical, Chemical, Microbiological, and Sensory Quality of Farmed Rainbow Trout (Oncorhynchus mykiss) Cultured in the Eastern Black Sea during Frozen Storage (−18 ± 1 °C)”. References Ahmadifard, N., Murueta, J. H. 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Marine Pollution Bulletin , 128 , 197-201. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 15 Mar, 2026 Reviewers invited by journal 26 Feb, 2026 Editor assigned by journal 26 Feb, 2026 Submission checks completed at journal 23 Feb, 2026 First submitted to journal 18 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8912550","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597723858,"identity":"91bb9358-e9cd-4421-9246-27b20d54c432","order_by":0,"name":"Neslihan AKYURT","email":"","orcid":"","institution":"Giresun University","correspondingAuthor":false,"prefix":"","firstName":"Neslihan","middleName":"","lastName":"AKYURT","suffix":""},{"id":597723859,"identity":"3376887a-be94-4c82-872d-b19c155e2596","order_by":1,"name":"Koray KORKMAZ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACCTB5AMr4wMAMogyI18I4A64lgUgtzDzEaJFs7334meeMTb7ktMOPP9tUWCc2sDdvk2D8cQ+nFmme48bSPDfSLGdLpxkY55xJT2zgOVYmwZBQjFOLnEQaUNuHwwZy0gkGyblthxMbJHLMgFpwuwyohfk3REv6h8OWIC3yb/BrkZZIYwM67LCBtHSOYTMj2BYe/Foke46xWc45k2YgOTunmLHnTLpxG09asUVCGm4tEsfbmG+8OWZjIHE7ffOHHxXWsv3shzfe+GCDWwsIMPEg89hABH4NwEj/QUDBKBgFo2AUjHAAAGseTgqcBWL9AAAAAElFTkSuQmCC","orcid":"","institution":"University of Ordu","correspondingAuthor":true,"prefix":"","firstName":"Koray","middleName":"","lastName":"KORKMAZ","suffix":""}],"badges":[],"createdAt":"2026-02-18 22:08:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8912550/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8912550/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103858112,"identity":"4ee5b4d8-7741-4d90-a223-4ad6243f844c","added_by":"auto","created_at":"2026-03-03 18:55:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":159242,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis showing the molecular weight distribution of peptide fractions in the Alcalase-derived Rapana venosa protein hydrolysate.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8912550/v1/173634129d56b1aa0aef0194.png"},{"id":104401354,"identity":"36e31d58-1ac4-4d8c-b9e7-f4243bfd3dd3","added_by":"auto","created_at":"2026-03-11 12:12:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":956015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8912550/v1/94171cea-86f3-448d-909d-839215b8c6cc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural and Functıonal Characterızatıon of an Alcalase-Derıved Proteın Hydrolysate from Rapana Venosa for Functıonal Food Applıcatıons With Elemental Safety Assessment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMarine-derived proteins have attracted increasing attention in the fields of food and nutrition in recent years owing to their high biological value, balanced amino acid profiles, and diverse functional properties. The growing global demand for protein, together with the need for sustainable and alternative raw materials, has necessitated the evaluation of marine organisms beyond conventional fish species. In this context, protein hydrolysates obtained through enzymatic hydrolysis have emerged as promising ingredients, as their enhanced solubility, bioavailability, and functional potential render them valuable not only from a nutritional standpoint but also for technological applications (Cunha and Pintado 2022).\u003c/p\u003e\n\u003cp\u003eProtein hydrolysates are produced through controlled enzymatic hydrolysis, during which protein chains are cleaved into shorter peptides and free amino acids, resulting in substantial modifications in digestibility and functional behavior. Numerous studies have reported that low-molecular-weight peptides are particularly advantageous with respect to functional properties, especially antioxidant activity. However, the magnitude and nature of these functional attributes are strongly influenced by the type of enzyme employed, hydrolysis conditions, and the structural characteristics of the raw material (Garc\u0026iacute;a-Moreno et al. 2014). Consequently, the assessment of each novel marine resource in the form of protein hydrolysates offers the potential to yield distinct structural and functional outcomes.\u003c/p\u003e\n\u003cp\u003eRapana venosa has attracted attention as an alternative marine protein source due to its benthic lifestyle, high protein content, and wide geographical distribution. Nevertheless, its close interaction with sediment has placed this species in a controversial position with regard to the accumulation of heavy metals and potentially toxic elements (PTEs). Numerous studies have reported variable levels of Cd, Pb, As, and Hg in different tissues of R. venosa, with Cd often detected at comparatively higher concentrations than other metals in certain samples (Gedik, 2018; \u0026Ouml;nel et al. 2025; Ryabushko et al. 2022; Zhelyazkov et al. 2018). In contrast, Hg and Pb have frequently been reported as either undetectable or present at concentrations below the maximum permissible limits defined by national and international regulations (Altuğ and G\u0026uuml;ler 2002; Levent and Oztekin 2016).\u003c/p\u003e\n\u003cp\u003ePrevious studies have further demonstrated that metal accumulation in R. venosa varies markedly depending on tissue type. While metabolically active tissues such as the operculum and hepatopancreas generally exhibit higher metal concentrations, the edible muscle tissue is often reported to contain lower levels (Alkan and Alkan 2023). However, several investigations have indicated that Cd and Pb concentrations in muscle tissue may exceed regulatory limits under certain conditions, raising concerns regarding potential public health risks associated with long-term consumption (Gedik 2018; \u0026Ouml;nel et al. 2025; Ryabushko et al. 2022). Risk assessment approaches based on target hazard quotient (THQ), total THQ, and metal pollution index (MPI) have generally suggested low acute risk, while emphasizing that chronic exposure to Cd and As warrants careful consideration (Alkan and Alkan 2023, 2021; Bat et al. 2023; Levent and Oztekin 2016).\u003c/p\u003e\n\u003cp\u003eDespite this extensive body of literature, the majority of existing studies have focused on evaluating heavy metal content in raw R. venosa tissues, whereas the influence of protein processing on the partitioning of metals into protein-rich fractions has received limited attention. Moreover, investigations into the functional properties of R. venosa-derived proteins have predominantly centered on hemolymph-derived antimicrobial compounds (Dolashka et al. 2011; Kirilova et al. 2024; Krumova et al. 2021), hemocyanin-derived components (Dolashka et al. 2016; Dolashka-Angelova et al. 2008), and gelatin-like products (Gaspar-Pintiliescu et al. 2019). In contrast, studies examining the edible muscle tissue\u0026mdash;which represents a protein-rich and widely consumed fraction\u0026mdash;as a functional ingredient remain scarce. This discrepancy underscores a significant knowledge gap regarding the functionalization of R. venosa muscle proteins.\u003c/p\u003e\n\u003cp\u003eAccordingly, the evaluation of protein hydrolysates not only as functional components but also as products with a more controlled elemental profile represents an important and underexplored research area. In particular, whether a marine species commonly associated with environmental risk concerns can be transformed into a functional protein source through enzymatic hydrolysis, while simultaneously addressing safety-related considerations, remains a critical question.\u003c/p\u003e\n\u003cp\u003eThe present study aims to comprehensively investigate the structural, functional, and safety-related characteristics of a protein hydrolysate obtained from Rapana venosa muscle using Alcalase. To this end, the degree of hydrolysis, amino acid composition, molecular weight distribution, color properties, antioxidant and antimicrobial activities, as well as elemental and heavy metal contents were evaluated. This work seeks to elucidate the potential of a protein-rich yet safety-challenged marine resource to be valorized as a functional protein component for food applications through enzymatic hydrolysis, while explicitly considering its associated safety profile.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMATERIAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRapana venosa muscle used as the research material was obtained from a marine snail processing and packaging facility operating in the Fatsa district of Ordu Province, T\u0026uuml;rkiye. The samples were divided into approximately 1 kg portions, placed in insulated polystyrene boxes containing ice, and transported to the Processing Technology Laboratory of the Department of Fisheries Technology Engineering, Faculty of Marine Sciences, Ordu University, under cold-chain conditions. Upon arrival, the samples were stored in frozen form at \u0026minus;20 \u0026deg;C until use in protein hydrolysate production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHydrolysate Production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen Rapana venosa muscle samples were thawed at room temperature prior to analysis, then minced and homogenized using a meat grinder (Empero E.M.P.12.01.P). To eliminate the potential contribution of endogenous enzymes to the hydrolysis process, the homogenized samples were heat-treated in a water bath at 90 \u0026deg;C for 20 min to ensure endogenous enzyme inactivation. After thermal treatment, the samples were cooled and diluted with distilled water at a ratio of 1:1 (w/v), followed by further homogenization to obtain a suitable matrix for enzymatic hydrolysis.\u003c/p\u003e\n\u003cp\u003eProtein hydrolysate production was carried out based on the enzymatic hydrolysis method optimized by Korkmaz (2018). A commercial alkaline protease, Alcalase, was used as the hydrolyzing enzyme. The optimum hydrolysis temperature, pH range, and enzyme inactivation conditions were applied in accordance with the technical specifications provided by the manufacturer. Accordingly, enzymatic hydrolysis was performed at 50 \u0026deg;C, and enzyme inactivation was achieved by heating at 85 \u0026deg;C for 10 min. In this study, hydrolysis conditions were applied based on previously optimized parameters targeting protein hydrolysis degree and process yield, rather than optimization toward a specific biological activity.\u003c/p\u003e\n\u003cp\u003eAccording to the method described by Korkmaz (2018), the homogenized samples were hydrolyzed at 50 \u0026deg;C using an enzyme concentration of 1% (w/w) and a hydrolysis time of 1 h. Following hydrolysis, enzymatic activity was terminated by placing the reaction mixtures in a water bath at 85 \u0026deg;C for 10 min, after which the samples were cooled at room temperature for 15 min. The cooled hydrolysate solutions were then centrifuged at 4000 rpm for 20 min to achieve phase separation. No distinct lipid phase was observed after centrifugation; instead, three fractions were obtained: an upper fraction containing minor protein\u0026ndash;lipid components, a middle aqueous fraction rich in protein, and a bottom fraction consisting of insoluble materials. The protein-rich middle fraction was used for the characterization and subsequent analyses of the Rapana venosa protein hydrolysate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximate Composition Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proximate composition of the protein hydrolysate was evaluated by determining crude protein, crude lipid, moisture, and ash contents. Crude protein content was determined based on total nitrogen using the Kjeldahl method (N \u0026times; 6.25) according to AOAC (1998). Moisture content was analyzed following AOAC (1990), and ash content was determined according to AOAC method 935.47 (1998).\u003c/p\u003e\n\u003cp\u003eTotal lipid content was determined using the method of Bligh and Dyer (1959). Briefly, 15 g of protein hydrolysate sample was homogenized, and 120 mL of a methanol/chloroform mixture (1:2, v/v) was added. The mixture was blended using a Waring blender, followed by the addition of 20 mL of 0.4% (w/v) CaCl₂ solution. The resulting suspension was then filtered through filter paper (Schleicher \u0026amp; Schuell, 595 1/2, 185 mm).\u003c/p\u003e\n\u003cp\u003eThe filtrate was transferred into pre-weighed round-bottom flasks, which were sealed to prevent solvent evaporation and stored in the dark overnight. After this period, the upper phase consisting of methanol and water was removed using a separatory funnel. The remaining chloroform phase was evaporated at 60 \u0026deg;C in a water bath using a rotary evaporator (Heidolph Basis Hei-Vap ML). The residual lipid fraction was further dried in an oven at 60 \u0026deg;C for 1 h to ensure complete removal of chloroform residues (Termal G11540SD). The flasks were then cooled to room temperature in a desiccator and weighed using an analytical balance with a sensitivity of 0.1 mg (Precisa XB 220A).\u003c/p\u003e\n\u003cp\u003eTotal lipid content was calculated using the following equation:\u003c/p\u003e\n\n\u003cp\u003eLipid content (%) = [(Weight of flask + lipid) \u0026minus; Weight of empty flask] \u0026times; 100 / Sample weight (g)\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDegree of Hydrolysis\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe degree of hydrolysis (DH, %) of the protein hydrolysate was determined using the trichloroacetic acid (TCA) solubility method described by Hoyle and Merritt (1994). This method is based on the solubility of low-molecular-weight peptides and free amino acids generated during enzymatic hydrolysis and is widely used for assessing the extent of protein hydrolysis in protein hydrolysates.\u003c/p\u003e\n\n\u003cp\u003eFollowing the completion of enzymatic hydrolysis, the hydrolysate was mixed with 20% (w/v) TCA solution at a ratio of 1:1 (v/v). The mixture was then centrifuged at 15,000 \u0026times; g for 20 min at 4 \u0026deg;C. After centrifugation, the clear supernatant containing proteins and peptides soluble in 10% TCA was collected, and all analyses were performed in triplicate.\u003c/p\u003e\n\u003cp\u003eThe degree of hydrolysis was calculated based on the ratio between the amount of protein soluble in 10% TCA and the total protein content of the hydrolysate. Total protein content was determined using the Kjeldahl method. The degree of hydrolysis was expressed according to the following equation:\u003c/p\u003e\n\n\u003cp\u003eDH (%) = (N₀ / Nᵀ) \u0026times; 100\u003c/p\u003e\n\n\u003cp\u003ewhere\u003c/p\u003e\n\u003cp\u003eN₀ represents the amount of protein soluble in 10% TCA, and\u003c/p\u003e\n\u003cp\u003eNᵀ represents the total protein content of the hydrolysate.\u003c/p\u003e\n\n\u003cp\u003eIn this study, the degree of hydrolysis was evaluated to assess the effectiveness of the applied hydrolysis conditions and the extent of protein conversion into low-molecular-weight fractions; no optimization targeting specific biological activity was conducted.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eElectrophoresis\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe molecular weight distribution of the protein hydrolysate was determined by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS-PAGE). The analyses were performed according to the discontinuous gel electrophoresis system described by Laemmli (1970).\u003c/p\u003e\n\n\u003cp\u003eFor this purpose, protein hydrolysate samples were diluted to an appropriate concentration and mixed with sample buffer containing SDS, glycerol, Tris\u0026ndash;HCl, and bromophenol blue. The samples were heated at 95 \u0026deg;C for 5 min to ensure complete protein denaturation. After cooling, the samples were loaded onto polyacrylamide gels consisting of a 4% stacking gel and a 12% resolving gel.\u003c/p\u003e\n\n\u003cp\u003eElectrophoresis was carried out under constant voltage conditions to allow separation of protein fractions based on their molecular weights. Upon completion of electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 to visualize protein bands and subsequently destained using an appropriate solvent to remove background staining.\u003c/p\u003e\n\n\u003cp\u003eThe molecular weights of the protein bands were estimated by comparison with a concurrently run protein molecular weight marker. SDS-PAGE analysis was employed to visually assess the conversion of proteins into lower-molecular-weight peptide fractions as a result of enzymatic hydrolysis.\u003c/p\u003e\n\n\n\n\n\n\u003cp\u003e\u003cstrong\u003eColor\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe color properties of the film samples obtained from the protein hydrolysate were determined using the CIE Lab* color space, where L* represents lightness, a* represents the red\u0026ndash;green axis, and b* represents the yellow\u0026ndash;blue axis. Color measurements were carried out using a Konica Minolta CM-5 colorimeter (Osaka, Japan).\u003c/p\u003e\n\n\u003cp\u003eMeasurements were performed to evaluate the homogeneity of the film surface and to assess the effect of the hydrolysis process on the visual characteristics of the samples. For each film sample, color values were recorded at multiple points on the surface, and the mean values were used for further analysis.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eHeavy Metal Determination\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe determination of heavy metals and mineral elements in the Rapana venosa protein hydrolysate was carried out using inductively coupled plasma mass spectrometry (ICP-MS). The analyses were performed through an accredited analytical laboratory specializing in elemental analysis.\u003c/p\u003e\n\n\u003cp\u003eMeasurements were conducted to determine the elemental composition and potential heavy metal content of the protein hydrolysate. Analytical results were calculated on a dry weight basis and expressed as mg/kg.\u003c/p\u003e\n\n\u003cp\u003eThe obtained data were used to evaluate the effect of the enzymatic hydrolysis process on the transfer of heavy metals potentially present in Rapana venosa muscle tissue into the hydrolysate fraction.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTotal and Free Amino Acid Analysis\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe total amino acid composition of the Rapana venosa protein hydrolysate was determined based on the methods reported by Lee and Hwang (2017) and Chan and Matanjun (2017), with appropriate modifications to adapt the procedure to the protein hydrolysate matrix. Amino acid profiling was performed using liquid chromatography\u0026ndash;tandem mass spectrometry (LC-MS/MS), which provides high sensitivity and selectivity (Thermo Fisher Scientific Inc., Waltham, MA, USA).\u003c/p\u003e\n\n\u003cp\u003eFor this purpose, 0.2 g of the protein hydrolysate sample was homogenized and weighed into a solution containing 10 mL of 6 N HCl. To prevent the loss of volatile components during hydrolysis, the samples were tightly sealed in test tubes, vortexed for 5 min, and hydrolyzed in an oven at 110 \u0026deg;C for 24 h to ensure complete cleavage of proteins into their constituent amino acids. After hydrolysis, the samples were cooled to room temperature and centrifuged at 4000 rpm for 15 min at 4 \u0026deg;C.\u003c/p\u003e\n\n\u003cp\u003eThe clear supernatant obtained after centrifugation was filtered through a 0.45 \u0026micro;m PTFE membrane filter to remove particulate matter and subsequently injected into the LC-MS/MS system for analysis. The resulting amino acid profile was used to evaluate the nutritional quality of the protein hydrolysate and to assess the free amino acids released as a result of enzymatic hydrolysis.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAntioxidant Activity\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe antioxidant activity of the protein hydrolysate was evaluated using two complementary methods based on different antioxidant mechanisms: CUPRAC (Cupric Ion Reducing Antioxidant Capacity) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assays. Prior to analysis, protein hydrolysate samples were dissolved in distilled water at a concentration of 1 mg/mL, and all measurements were performed in triplicate.\u003c/p\u003e\n\n\u003cp\u003eFor the CUPRAC assay, the reagent was freshly prepared by mixing equal volumes of 10 mM CuCl₂, 7.5 mM neocuproine (prepared in ethanol), and 1 M ammonium acetate buffer (pH 7.0). The hydrolysate solution was mixed with the CUPRAC reagent, and the reaction mixture was incubated at room temperature for 30 min in the dark. After incubation, absorbance values were measured at 450 nm using a UV\u0026ndash;Vis spectrophotometer. Antioxidant capacity was calculated based on a Trolox standard curve and expressed as \u0026micro;mol Trolox equivalents (TE)/g hydrolysate.\u003c/p\u003e\n\n\u003cp\u003eDPPH radical scavenging activity was determined using a 0.1 mM DPPH solution prepared in methanol. The hydrolysate solution was mixed with the DPPH solution to a final volume of 1.0 mL and vortexed thoroughly. The mixture was incubated at room temperature for 30 min under dark conditions. The decrease in absorbance of the purple DPPH radical, resulting from its interaction with antioxidant components in the protein hydrolysate, was measured at 517 nm using a UV\u0026ndash;Vis spectrophotometer. DPPH radical scavenging activity was calculated based on the reduction in absorbance relative to the control, and the results were expressed as \u0026micro;mol TE/g hydrolysate using a Trolox standard curve.\u003c/p\u003e\n\n\u003cp\u003eThe antimicrobial activity of the protein hydrolysates was evaluated using the agar well diffusion method against two reference bacterial strains: one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Bacterial cultures were activated on Mueller\u0026ndash;Hinton Agar (MHA) and adjusted to the 0.5 McFarland standard (approximately 1 \u0026times; 10⁸ CFU/mL). The prepared bacterial suspensions were uniformly spread onto the surface of MHA plates using sterile cotton swabs.\u003c/p\u003e\n\n\u003cp\u003eTo prevent microbial contamination, protein hydrolysate solutions were sterilized by filtration through 0.22 \u0026micro;m pore-size membrane filters. The final concentrations used in the assay were prepared in the range of 25\u0026ndash;50 mg/mL. Wells with a diameter of 6 mm were aseptically punched into the agar surface, and 50\u0026ndash;100 \u0026micro;L of the hydrolysate solutions were added to each well. To allow diffusion of the hydrolysates into the agar medium, the plates were kept at room temperature for 30 min prior to incubation, followed by incubation at 37 \u0026deg;C for 24 h.\u003c/p\u003e\n\n\u003cp\u003eAfter incubation, clear zones indicating bacterial growth inhibition were measured using a digital caliper, and antimicrobial activity was recorded as inhibition zone diameter (mm). Ampicillin (10 \u0026micro;g) was used as a positive control, while the solvent used for dissolving the hydrolysates (sterile water or 50% ethanol) served as the negative control. The antimicrobial assay was conducted to delineate the potential biological activity boundaries of the protein hydrolysate.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eProxymate Composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hydrolysate exhibited a protein-rich composition with a high protein content (75.5%) and a low lipid level (3.4%), indicating a matrix enriched in protein while being limited in lipid fraction. This composition suggests that the applied enzymatic hydrolysis and subsequent fractionation steps were effective in enriching the protein fraction.\u003c/p\u003e\n\u003cp\u003eThe relatively high ash content (12.6%) can be attributed to the characteristic mineral composition of marine-derived raw materials. Similarly, the literature reports that ash content in various marine protein hydrolysates may vary over a wide range, depending on the species, tissue type, and processing conditions applied (Henriques et al. 2021).\u003c/p\u003e\n\u003cp\u003eBased on these proximate composition findings, the predominance of the protein fraction indicates that the matrix provides a quantitatively substantial protein source from which short-chain peptides and free amino acids associated with functional activity may be released during enzymatic hydrolysis. The extent to which this potential is realized is elucidated through the evaluation of post-hydrolysis structural transformations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDegree of Hydrolysis (%HD)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, the degree of hydrolysis of the protein hydrolysate obtained from Rapana venosa muscle was determined to be 55.5%. This value indicates that peptide bonds within the protein chains were extensively cleaved during enzymatic hydrolysis, reflecting a high level of enzymatic breakdown. The degree of hydrolysis is considered a critical parameter in the production of protein hydrolysates, as it represents a fundamental indicator of the extent of peptide bond cleavage from the parent protein (Benjakul and Morrissey 1997). The percentage degree of hydrolysis (%DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate and is widely accepted as one of the most important measures of hydrolysis efficiency.\u003c/p\u003e\n\u003cp\u003eIt has been reported in the literature that an increase in the degree of hydrolysis is associated with enhanced protein recovery, the formation of lower molecular weight peptides due to protein chain fragmentation, and a consequent improvement in water solubility, ultimately leading to enhanced functional properties of the hydrolysate (Shahidi et al. 1995).\u003c/p\u003e\n\u003cp\u003eAlcalase has been widely reported to achieve higher degrees of hydrolysis compared to other commercial proteases. Ovissipour et al. (2012) investigated the hydrolysis of Persian sturgeon proteins using five different enzymes (Alcalase, Protamex, Neutrase, Flavourzyme, and Trypsin) and reported that the highest degree of hydrolysis was obtained with Alcalase. Similarly, Yoon et al. (2015) hydrolyzed liver extracts from Oncorhynchus keta and Oncorhynchus gorbuscha using various commercial enzymes and demonstrated that the highest soluble protein recovery was achieved with Alcalase at 50 °C and pH 7.0. In a comparative study conducted on rice bran protein concentrate and soy protein, Ahmadifard et al. (2016) also reported that Alcalase exhibited a higher hydrolytic capacity than other enzymes.\u003c/p\u003e\n\u003cp\u003eOverall, the literature indicates that Alcalase typically produces degrees of hydrolysis ranging from approximately 16% to 67% in proteins of aquatic origin (Edison et al. 2020; Mohanty et al. 2021; Pires et al. 2024; Rajabzadeh et al. 2018; Sierra-Lopera et all. 2021). This behavior is attributed to the enzyme’s high catalytic efficiency and broad substrate specificity. The 55.5% degree of hydrolysis determined for Rapana venosa in the present study falls within the upper range of reported values, indicating efficient protein cleavage and suggesting that an appropriate hydrolysis level was achieved for the generation of biologically valuable low-molecular-weight peptide fractions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal and Free Amino acids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino acid composition of the protein hydrolysate obtained from Rapana venosa muscle was evaluated by separately determining total and free amino acid fractions. This approach enabled the assessment of both the overall nutritional profile and the extent to which enzymatic hydrolysis promoted the release of amino acids into the free form. The results are summarized in Table 1.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmino Acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTotal AA (mg/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFree AA (mg/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAlanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGlycine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eThreonine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLysine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eArginine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHistidine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAspartic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCystine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGlutamic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSerine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHydroxyproline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eValine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMethionine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLeucine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIsoleucine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAsparagine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePhenylalanine*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* Essential amino acids (EAA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Amino acid composition of the protein hydrolysate obtained from Rapana venosa muscle, expressed as total and free amino acid fractions (mg/g).\u003c/p\u003e\n\u003cp\u003eThe predominance of glutamic acid and aspartic acid in both total and free amino acid distributions demonstrates that the hydrolysate is rich in amino acids bearing acidic side chains. The presence of these amino acids at high levels in the free fraction suggests that the post-hydrolysis structure is highly exposed to radical interactions. It has been reported that acidic amino acids make a strong contribution to free radical scavenging activity, while exhibiting limited involvement in metal-reduction–based antioxidant mechanisms (Udenigwe and Aluko 2011).\u003c/p\u003e\n\u003cp\u003eWith respect to basic amino acids, lysine and arginine were detected at considerable levels in both the total and free fractions. Owing to the charged groups present in their side chains, these amino acids are capable of directly interacting with free radicals and forming complexes with metal ions (Aissaoui et al. 2017; Hu et al. 2020). The notable presence of lysine in the free amino acid fraction suggests that the hydrolysate acquires enhanced molecular reactivity and functional potential following enzymatic hydrolysis.\u003c/p\u003e\n\u003cp\u003eAromatic amino acids, namely phenylalanine and tyrosine, were identified in both total and free amino acid profiles, supporting their contribution to the radical scavenging behavior of the hydrolysate. Aromatic side chains are known to donate protons to electron-deficient radicals, rendering these amino acids key contributors to free radical quenching mechanisms (Rajapakse et al. 2005; Yang et al. 2019). Their occurrence in the free fraction further indicates that antioxidant activity is not merely related to total protein content, but is strongly influenced by molecular accessibility achieved through hydrolysis.\u003c/p\u003e\n\u003cp\u003eAmong aliphatic amino acids, leucine, valine, and alanine were found at elevated levels in the free fraction, a result that is consistent with the characteristic proteolytic specificity of Alcalase. It has been reported that Alcalase effectively cleaves peptide bonds adjacent to aliphatic and aromatic amino acids, generating hydrolysates with enhanced biological activities, including antioxidant properties (Paek et al. 2001; Qian et al. 2008). Accordingly, the amino acid distribution observed in the present study reflects a structured enzymatic transformation driven by enzyme–substrate interactions rather than a nonspecific degradation process.\u003c/p\u003e\n\u003cp\u003eThe relatively low levels of sulfur-containing amino acids, such as cysteine and methionine, suggest that the antioxidant behavior of the hydrolysate is predominantly governed by acidic, basic, and aromatic amino acids. Although the thiol group of cysteine is known to exhibit strong antioxidant activity (Qian et al. 2008), the present findings indicate that the antioxidant response of the hydrolysate does not rely primarily on sulfur-containing residues.\u003c/p\u003e\n\u003cp\u003eIt is well established that the antioxidant properties of peptides are closely related to their amino acid composition, molecular structure, and hydrophobicity (Alemán et al. 2011; Chen et al. 1998). When the high abundance of free amino acids is considered alongside the dominance of low-molecular-weight fractions, the antioxidant behavior of the Rapana venosa protein hydrolysate can be interpreted as a direct consequence of increased molecular accessibility and reactivity resulting from enzymatic hydrolysis. Collectively, these findings demonstrate that the antioxidant activity of the hydrolysate can be coherently explained within a composition-driven and structure–activity–consistent framework.\u003c/p\u003e\n\u003cp\u003eWhen the total and free amino acid profiles of the Rapana venosa protein hydrolysate are evaluated together, it becomes evident that the observed antioxidant behavior is not a random outcome, but rather is closely associated with the amino acid composition and the structural transformations induced by enzymatic hydrolysis. In particular, the pronounced enrichment of specific amino acids in the free fraction indicates that hydrolysis effectively occurred along the protein backbone, rendering functional building blocks accessible within the matrix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-Page\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSDS-PAGE analysis was applied to the protein hydrolysate in order to elucidate the presence of peptide fractions formed in accordance with the determined degree of hydrolysis and to assess their molecular weight distribution. Examination of the electrophoretic profile obtained from SDS-PAGE analysis revealed a pronounced reduction in the bands corresponding to 25 and 70 kDa, accompanied by an evident increase in band intensity within the low-molecular-weight fractions. In particular, the predominance of bands below 25 kDa indicates that the applied enzymatic hydrolysis process resulted in an advanced level of protein chain cleavage.\u003c/p\u003e\n\u003cp\u003eIn the literature, a decrease in high-molecular-weight bands and the predominance of low-molecular-weight bands in SDS-PAGE profiles of protein hydrolysates are widely accepted as key indicators of increased degree of hydrolysis. The appearance of intense banding within the 10–25 kDa range or below following enzymatic hydrolysis has been reported as a characteristic outcome of successful and advanced hydrolysis processes (Zaky et al. 2019). In this context, the predominance of low-molecular-weight bands observed in the Rapana venosa protein hydrolysate is structurally consistent with the determined degree of hydrolysis of 55.5%.\u003c/p\u003e\n\u003cp\u003eBui et al. (2021) reported that bioactive peptides generally consist of 3–20 amino acid residues and predominantly possess molecular weights below 50 kDa. Raghavan and Kristinsson (2008) demonstrated that tilapia protein hydrolysates with molecular weights in the range of 3.5–10 kDa exhibited stronger antioxidant activity. Silvestre et al. (2013) classified the relationship between degree of hydrolysis and peptide size into four levels, indicating that hydrolysates with degrees of hydrolysis between 20% and 50% predominantly contain peptides smaller than 3 kDa and that this segment represents a suitable range for the generation of bioactive peptides. This classification is directly consistent with the 55.5% degree of hydrolysis determined in the present study.\u003c/p\u003e\n\u003cp\u003eAccordingly, evaluation of the RVPH indicates that high-molecular-weight protein structures were largely eliminated, while low-molecular-weight peptide fractions—widely regarded as more functional in both biological and technological contexts—became predominant. This structural transformation provides a coherent and robust framework for interpreting the functional behavior of the hydrolysate at the molecular level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColor Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe color parameters of the Rapana venosa protein hydrolysate were determined as L* = 88.01, a* = −1.62, and b* = 15.16. These values indicate that the hydrolysate exhibits a light-colored appearance with high brightness and minimal chromatic contribution. The high L* value reflects a visually light and homogeneous product, while the negative a* value confirms the absence of red tonal components. The moderate b* value suggests a limited degree of yellow coloration.\u003c/p\u003e\n\u003cp\u003eIt has been reported that the color characteristics of protein hydrolysates vary widely depending on the raw material and the tissue source used. Thiansilakul et al. (2007) reported L* = 58.00, a* = 8.38, and b* = 28.3 for round scad protein hydrolysate, corresponding to a pronounced brownish–yellow color profile. Similarly, Kumari et al. (2023) determined L* = 57.1 ± 1.5, a* = 3.0 ± 0.1, and b* = 10.9 ± 0.5 for pink perch viscera protein hydrolysate powder, describing the product as dark with brownish–yellow tones. In the same study, head-derived protein hydrolysates exhibited higher L* and lower a* and b* values compared to viscera-derived samples. Consistently, Hassan et al. (2019) emphasized that protein hydrolysates obtained from visceral tissues generally display darker color profiles.\u003c/p\u003e\n\u003cp\u003eThe yellow–brown coloration commonly observed in protein hydrolysates has been largely attributed to intermediate or advanced Maillard reaction products. In this context, decreases in L* values accompanied by increases in a* and b* values are often associated with the presence of reactive proteins and free amino acids involved in non-enzymatic browning reactions (Kumari et al. 2023; Liu et al. 2004). In the present study, the simultaneous occurrence of a high L* value and a negative a* value indicates the absence of noticeable Maillard-related browning, suggesting that color-related chemical stability was preserved during processing. This finding distinguishes the Rapana venosa protein hydrolysate from many previously reported marine protein hydrolysates characterized by darker coloration.\u003c/p\u003e\n\u003cp\u003eFurthermore, light-colored protein hydrolysates have been reported to exhibit higher visual acceptability compared to darker counterparts (Kumari et al. 2023). From this perspective, the neutral and light color profile of the Rapana venosa protein hydrolysate may be considered advantageous for its potential application in functional food formulations and protein-based industrial products where visual quality is an important factor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntioxidant Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the antioxidant capacity of the Rapana venosa protein hydrolysate was evaluated using two complementary assays based on different mechanisms of action: DPPH radical scavenging activity and CUPRAC (Cu²⁺ reducing capacity). It is well established that the antioxidant potential of protein hydrolysates and bioactive peptides is not restricted to a single mechanism, but may arise through multiple pathways, including free radical scavenging, hydrogen donation, and modulation of redox processes (Frankel and Meyer 2000). Therefore, the combined application of these two methods enables a mechanism-oriented evaluation of the antioxidant behavior of the hydrolysate.\u003c/p\u003e\n\u003cp\u003eThe DPPH assay demonstrated that the Rapana venosa protein hydrolysate exhibited a radical inhibition of 55.17% at a concentration of 1 mg/mL. This level of activity is higher than, or comparable to the upper range of, values reported for many marine protein hydrolysates, indicating a pronounced free radical scavenging capacity. Previous studies have shown that DPPH radical scavenging activities of protein hydrolysates derived from various fish and plant sources generally range between 15% and 45% (García-Moreno et al. 2014). Jamdar et al. (2010) and Wu et al. (2003) further reported that increases in the degree of hydrolysis enhance DPPH scavenging activity, primarily due to the generation of low-molecular-weight peptides and free amino acids. In this context, the relatively high inhibition observed in the present study suggests that the applied enzymatic hydrolysis conditions effectively promoted the release of antioxidant-active peptide fractions.\u003c/p\u003e\n\u003cp\u003eWhen the DPPH results are evaluated together with amino acid composition and SDS-PAGE findings, a clear mechanistic explanation emerges. The hydrolysate was characterized by a predominance of low-molecular-weight peptide fractions and an amino acid profile enriched in electron-donating amino acids, including glutamic acid, aspartic acid, alanine, and glycine. These structural features are known to facilitate hydrogen donation and direct interactions with free radicals, thereby contributing to the stabilization of reactive species and the termination of radical chain reactions. Accordingly, the strong radical scavenging activity observed in the DPPH assay can be directly attributed to the peptide composition and molecular size distribution of the hydrolysate.\u003c/p\u003e\n\u003cp\u003eIn contrast, no notable reducing capacity was detected in the CUPRAC assay. This discrepancy between DPPH and CUPRAC results can be explained by the distinct antioxidant mechanisms underlying these methods. While the DPPH assay primarily reflects hydrogen donation and free radical scavenging ability, the CUPRAC method is based on the reduction of metal ions. Therefore, the absence of CUPRAC activity does not contradict the DPPH findings; rather, it indicates that the antioxidant behavior of the Rapana venosa protein hydrolysate is not associated with metal ion reduction. Instead, the results demonstrate a mechanism-specific antioxidant profile in which the antioxidant potential is predominantly governed by radical scavenging pathways linked to the structural characteristics of the peptide fractions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial activity of the protein hydrolysate obtained from Rapana venosa muscle was evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. The results demonstrated that the hydrolysate did not exhibit inhibitory activity against either of the tested bacterial strains.\u003c/p\u003e\n\u003cp\u003ePrevious studies have consistently reported that antimicrobial activity in mollusks is primarily associated with hemolymph-derived proteins and peptides rather than muscle tissue. In particular, hemocyanins and hemocyanin-derived peptides present in the hemolymph have been shown to display agglutinative and antibacterial properties against a range of pathogenic microorganisms (Dolashka et al. 2011; Krilova et al. 2024). In contrast, there is limited evidence in the literature supporting a comparable antimicrobial defense function for muscle tissue or edible flesh fractions.\u003c/p\u003e\n\u003cp\u003eIn this context, the absence of antimicrobial activity observed for the Rapana venosa muscle protein hydrolysate is fully consistent with the established understanding that antimicrobial defense mechanisms in mollusks are predominantly mediated by immune-related hemolymph components rather than structural tissues. Therefore, the lack of antimicrobial effect should not be regarded as a limitation of the hydrolysate, but rather as a biologically coherent outcome aligned with the tissue origin of the raw material and the current body of literature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElemental Composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Elemental composition of Rapana venosa muscle reported in the literature and the protein hydrolysate obtained in the present study (dry weight basis).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eElement\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRapana venosa muscle (literature range, mg/kg, dry weight)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eProtein hydrolysate (this study, mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2 – 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1 – 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5 – 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1 – 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5 – 25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5 – 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003etrace\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1 – 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003etrace\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e150 – 450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e172.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eZn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40 – 120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 – 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e800 – 3000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12,546.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData compiled from multiple literature sources (e.g., Alkan and Alkan, 2023; Altuğ and Güler 2002; Bayraklı et al. 2024; Gedik 2018; Levent and Öztekin, 2016; Mülayim and Balkıs 2015; Önel et al., 2025; Ryabushko et al. 2022; Zhelyazkov et al. 2018). The literature ranges represent the minimum and maximum concentrations reported for Rapana venosa muscle tissue across different studies and reflect variability associated with habitat characteristics, sampling location, and analytical methods.\u003c/p\u003e\n\u003cp\u003eThe heavy metal and mineral element contents of Rapana venosa meat and the protein hydrolysate obtained in this study are comparatively presented in Table 2. In the literature, heavy metal concentrations reported for Rapana venosa muscle tissue are known to vary widely, largely due to the benthic lifestyle of the species and its close interaction with sediment. In particular, potentially toxic elements such as Cd, Pb, As, and Hg have been reported at variable levels in raw tissues across different studies.\u003c/p\u003e\n\u003cp\u003eIn the present study, Cr and Cd were not detected (ND) in the protein hydrolysate, while Ni and Co were detected only at trace levels. In contrast, the concentrations of Pb (0.80 mg/kg), Hg (0.33 mg/kg), and As (15.57 mg/kg) were within, or close to the lower bounds of, the ranges previously reported for Rapana venosa meat in the literature. These findings suggest that the enzymatic hydrolysis process, together with subsequent centrifugation and fractionation steps, may limit the transfer of certain heavy metals into the protein-rich hydrolysate fraction.\u003c/p\u003e\n\u003cp\u003eThe absence of detectable Cd in the hydrolysate fraction is particularly noteworthy. Although Cd has been reported at relatively high levels in Rapana venosa tissues in some studies, its non-detectable level in the protein hydrolysate indicates that metal–protein interactions may exhibit tissue- and fraction-specific behavior. Similarly, the non-detection of Cr in the hydrolysate fraction suggests that a substantial portion of this element may remain associated with insoluble residues or fractions separated during the hydrolysis process.\u003c/p\u003e\n\u003cp\u003eComparable observations have been reported for protein hydrolysates derived from other marine species. Mangano et al. (2021) investigated the heavy metal content of anchovy protein hydrolysates and reported that the levels detected were below maximum limits. Similarly, la Fuente et al. (2023) examined protein hydrolysates obtained from mackerel and salmon and concluded that their heavy metal contents remained within acceptable safety ranges. In a broader evaluation, Wu et al. (2022) emphasized that fish protein hydrolysates are generally not considered a potential risk in terms of heavy metal content. These findings support the view that enzymatic hydrolysis may influence the distribution and bioavailability of metals within protein-rich fractions across different marine species.\u003c/p\u003e\n\u003cp\u003eWith respect to nutritionally essential elements, Fe (172.30 mg/kg), Zn (40.29 mg/kg), Cu (27.73 mg/kg), and especially Mg (12,546.94 mg/kg) were retained at appreciable levels in the hydrolysate fraction. This observation indicates that while the enzymatic hydrolysis process may restrict the migration of certain potentially toxic elements, it allows nutritionally valuable minerals to be preserved within the protein-rich fraction.\u003c/p\u003e\n\u003cp\u003eTaken together, these results demonstrate that a marine resource such as Rapana venosa, which is often discussed in the literature in terms of heavy metal accumulation, can exhibit a more controlled and predictable elemental profile when processed into a protein hydrolysate via enzymatic hydrolysis. These findings highlight the importance of evaluating protein hydrolysates not only in terms of their functional properties but also with respect to their elemental safety profiles, and suggest that enzymatic hydrolysis may represent a promising processing strategy for the utilization of environmentally sensitive marine protein sources.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a protein hydrolysate was produced from Rapana venosa muscle tissue using Alcalase, and the resulting product was comprehensively evaluated in terms of proximate composition, degree of hydrolysis, amino acid profile, molecular weight distribution, color characteristics, antioxidant capacity, antimicrobial activity, and elemental/heavy metal content. The findings collectively demonstrate that the hydrolysate is not merely a protein-rich product, but rather exhibits a coherent and predictable profile in terms of composition\u0026ndash;structure\u0026ndash;function relationships.\u003c/p\u003e\n\n\u003cp\u003eProximate analyses revealed that the hydrolysate possessed a high protein content (75.5%) and a low lipid level (3.4%), indicating a distinctly protein-based matrix. The relatively high ash content (12.6%) is consistent with the characteristic mineral composition of marine-derived raw materials and aligns well with values reported for comparable marine protein hydrolysates in the literature. This fundamental composition indicates the presence of a quantitatively dominant protein source from which short-chain peptides and free amino acids associated with functional activity can be released during enzymatic hydrolysis.\u003c/p\u003e\n\n\u003cp\u003eThe degree of hydrolysis was determined to be 55.5%, demonstrating that the applied enzymatic treatment resulted in an advanced level of protein chain cleavage. This finding was structurally confirmed by SDS-PAGE analysis, which showed a pronounced reduction in high-molecular-weight bands and the predominance of bands below 25 kDa. According to the literature, this hydrolysis range represents a favorable segment for the generation of biologically active low-molecular-weight peptides. Accordingly, the molecular weight distribution of the obtained hydrolysate indicates that the structural prerequisites for functional activity were successfully established.\u003c/p\u003e\n\n\u003cp\u003eTotal and free amino acid analyses demonstrated that the hydrolysate contained essential amino acids contributing to nutritional quality, while also exhibiting a composition enriched in glutamic and aspartic acids, conferring sensory and technological advantages. The marked presence of the free amino acid fraction confirms that the hydrolysis process was not merely nominal, but effectively progressed at the molecular level, rendering functionally relevant chemical groups accessible within the matrix. This enables the functional behavior of the hydrolysate to be interpreted directly on the basis of experimentally determined compositional and structural data.\u003c/p\u003e\n\n\u003cp\u003eColor measurements indicated that the hydrolysate exhibited a light and neutral appearance, characterized by a high L* value (88.01), a negative a* value (\u0026minus;1.62), and a moderate b* value (15.16). These results indicate the absence of pronounced Maillard-related browning during processing and suggest that the initial chemical stability of the product was preserved. The light color profile may therefore be considered advantageous for applications in functional food formulations and protein-based ingredient systems where visual acceptability is an important criterion.\u003c/p\u003e\n\n\u003cp\u003eFunctional evaluation revealed that the hydrolysate exhibited a DPPH radical inhibition of 55.17%, indicating a pronounced free radical scavenging capacity. In contrast, no significant reducing capacity was detected in the CUPRAC assay. This discrepancy demonstrates that the antioxidant behavior of the hydrolysate is governed primarily by radical scavenging and hydrogen-donating mechanisms rather than metal ion reduction. When considered together with the amino acid composition and the predominance of low-molecular-weight peptide fractions, the observed antioxidant activity can be explained within a composition-driven, mechanism-specific structure\u0026ndash;activity framework.\u003c/p\u003e\n\n\u003cp\u003eNo antimicrobial activity was observed against Staphylococcus aureus or Escherichia coli. Given that antimicrobial defense mechanisms in mollusks are predominantly associated with hemolymph-derived components rather than muscle tissue, the absence of antimicrobial activity in the muscle-derived protein hydrolysate is biologically coherent and consistent with existing literature.\u003c/p\u003e\n\n\u003cp\u003eOne of the most notable outcomes of this study concerns the elemental and heavy metal profile of the hydrolysate. The non-detection of Cr and Cd, the presence of Pb and Hg at low levels, and the retention of essential minerals such as Mg, Fe, Zn, and Cu indicate that enzymatic hydrolysis and fractionation steps can limit the transfer of potentially toxic elements into the protein-rich fraction while preserving nutritionally valuable minerals. These findings suggest that a marine resource such as Rapana venosa, which is often discussed in the literature in relation to heavy metal accumulation, can be converted\u0026mdash;under appropriate processing conditions\u0026mdash;into a protein hydrolysate with a more controlled and predictable elemental profile.\u003c/p\u003e\n\n\u003cp\u003eOverall, the results of this study demonstrate that the functional properties of Rapana venosa protein hydrolysate are not attributable to isolated analytical parameters, but rather emerge from the integrated effects of hydrolysis degree, molecular weight distribution, amino acid composition, and antioxidant behavior. Importantly, these functional outcomes are supported directly by experimentally measurable structural and compositional indicators, without reliance on theoretical assumptions or computational predictions.\u003c/p\u003e\n\n\u003cp\u003eIn conclusion, Rapana venosa protein hydrolysate represents a strong and multidimensional candidate for functional food formulations and protein-based ingredient applications, owing to its advanced hydrolysis characteristics, accessible and balanced amino acid composition, favorable color and stability profile, mechanism-specific antioxidant capacity, and improved heavy metal safety attributes. Future studies focusing on molecular-weight-based peptide fractionation and LC\u0026ndash;MS/MS-driven peptidomic identification, as well as in vitro digestion and bioaccessibility assessments, would further strengthen the application potential of this hydrolysate.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eK.K.: Methodology, Software (statistical analyses), Writing\u0026mdash;original draft, Investigation. N.A: Conceptualization, Writing, Methodology. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data generated and analyzed during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e \u003ca id=\"_anchor_1\" href=\"#_msocom_1\" language=\"JavaScript\" name=\"_msoanchor_1\"\u003e\u003c/a\u003e Not applicable, as this study did not involve human participants or clinical interventions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003eThis study is derived from a part of the PhD thesis entitled \u0026ldquo;Investigation of the Effects of Chitosan-Based Edible Films Enriched with Rapana venosa Protein Hydrolysate on the Physical, Chemical, Microbiological, and Sensory Quality of Farmed Rainbow Trout (Oncorhynchus mykiss) Cultured in the Eastern Black Sea during Frozen Storage (\u0026minus;18 \u0026plusmn; 1 \u0026deg;C)\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmadifard, N., Murueta, J. 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Journal of Food Measurement and Characterization, 13(4), 2542-2548.\u003c/li\u003e\n\u003cli\u003eZhelyazkov, G., Yankovska-Stefanova, T., Mineva, E., Stratev, D., Vashin, I., Dospatliev, L., ... \u0026amp; Popova, T. (2018). Risk assessment of some heavy metals in mussels (Mytilus galloprovincialis) and veined rapa whelks (Rapana venosa) for human health. \u003cem\u003eMarine Pollution Bulletin\u003c/em\u003e, \u003cem\u003e128\u003c/em\u003e, 197-201.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"thalassas-an-international-journal-of-marine-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thal","sideBox":"Learn more about [Thalassas: An International Journal of Marine Sciences](http://link.springer.com/journal/41208)","snPcode":"41208","submissionUrl":"https://submission.nature.com/new-submission/41208/3","title":"Thalassas: An International Journal of Marine Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rapana venosa, Protein hydrolysate, Enzymatic hydrolysis, Alcalase, Antioxidant activity, Heavy metals","lastPublishedDoi":"10.21203/rs.3.rs-8912550/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8912550/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRapana venosa is a benthic marine gastropod with high protein content; however, its use as a food-derived protein source is limited by concerns regarding potentially toxic elements. In this study, a protein hydrolysate was produced from R. venosa muscle using Alcalase and characterized in terms of structural, functional, and elemental properties. The hydrolysate showed a high degree of hydrolysis (55.5%), supported by amino acid profiling and SDS-PAGE analysis indicating the predominance of low-molecular-weight peptides. The amino acid composition revealed a substantial proportion of essential and functional amino acids, with glutamic and aspartic acids as dominant components.\u003c/p\u003e\n\u003cp\u003eThe hydrolysate exhibited notable antioxidant activity, achieving 55.17% DPPH radical scavenging at 1 mg/mL. No significant reducing capacity was observed in the CUPRAC assay, suggesting that antioxidant activity is mainly associated with radical scavenging rather than metal ion reduction. No antimicrobial activity was detected against Staphylococcus aureus or Escherichia coli, consistent with the association of antimicrobial defense in mollusks with hemolymph-derived components rather than muscle tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElemental analysis indicated the absence of chromium and cadmium, while lead and mercury were present at low levels. Nutritionally relevant minerals, including magnesium, iron, zinc, and copper, were retained in the protein-rich fraction. These findings demonstrate that enzymatic hydrolysis of R. venosa muscle yields a structurally and functionally favorable protein ingredient with a controlled elemental profile.\u003c/p\u003e","manuscriptTitle":"Structural and Functıonal Characterızatıon of an Alcalase-Derıved Proteın Hydrolysate from Rapana Venosa for Functıonal Food Applıcatıons With Elemental Safety Assessment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 18:54:49","doi":"10.21203/rs.3.rs-8912550/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"121014323060334853572658990514026131905","date":"2026-03-16T01:51:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-26T18:06:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T18:05:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T14:52:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Thalassas: An International Journal of Marine Sciences","date":"2026-02-18T22:02:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"thalassas-an-international-journal-of-marine-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thal","sideBox":"Learn more about [Thalassas: An International Journal of Marine Sciences](http://link.springer.com/journal/41208)","snPcode":"41208","submissionUrl":"https://submission.nature.com/new-submission/41208/3","title":"Thalassas: An International Journal of Marine Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a4791f0b-07ee-4934-ba5a-52b8ad88790a","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T18:54:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 18:54:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8912550","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8912550","identity":"rs-8912550","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}