A cross-sectional study design was employed to assess the biochemical composition and nutritional quality of commercially important shellfish species collected from Karachi Fish Harbor, Pakistan. This design was selected to allow systematic sampling and laboratory analysis of multiple species collected under uniform post-harvest and handling conditions within a defined time frame. Karachi Fish Harbor serves as the principal commercial landing and distribution hub for marine fisheries and receives catches from Pakistan's coastal waters. Therefore, sampling from this location ensures consistency, logistical feasibility, and representativeness of export-grade shellfish entering the national seafood supply chain.

Different specimens of export-quality shrimps (Penaeus monodon and Penaeus pulchricaudatus), lobsters (Panulirus ornatus and Thenus indicus), cuttlefish (Sepia pharaonis), and squid (Uroteuthis duvaceulii) were purchased from Karachi Fish Harbor, between April 2023 and June 2024, covering seasonal variability in commercial landings. Samples were purchased immediately after landing from authorized vendors to ensure freshness and minimize post-harvest deterioration. Freshly harvested samples (of a maximum 24–48 h) were transported to the laboratory under iced conditions.

The number of individuals (N) for each species varied due to availability (Table 1). Samples of each species were washed and cleaned properly. The specimen size in terms of length, width, and whole weight was measured and recorded before sample preparation. The edible muscle tissues were carefully extracted using sterile scalpels. The samples were homogenized using a laboratory-grade homogenizer (IKA T25 Digital Ultra-Turrax, Germany) under chilled conditions to prevent lipid oxidation. The prepared samples were then labeled and packed in a zip-lock bag and stored at −20 °C till analysis. Each analysis was performed in triplicate (n = 3) for statistical reliability.

All the extracted samples were first subjected to biometrical analysis to calculate percentage edibility (PE) and condition index (CI). The measurements were done according to the formula described by Mohite et al. (2009) and Okumus and Stirling (1998), respectively.

The proximate composition was determined by using Association of Official Analytical Chemists (AOAC, 2023) International standard methods 925.10, 923.03, and 960.52 for moisture, ash, and protein, respectively. For crude fat, the lipid extraction method suggested by Bligh and Dyer (1959) was used, while for energy value, the conversion factors 4 kcal/g for protein, 9 kcal/g for fat were used (Greenfield and Southgate, 2003).

Normal hydrolysis was carried out by the method described in AOAC (2023). Approximately 100 mg of each sample was subjected to hydrolysis by adding hydrochloric acid-phenol solution (6 N; 50 ml) in digestion tubes and placed under vacuum for 24 h at 110 °C. Washed the hydrolyzed samples with water and dried under vacuum by rotary evaporator, at 70 °C. The residue was reconstituted with deionized water and brought to a final volume of 25 ml, filtered, and appropriately diluted with buffer solution in a sample vial before being injected into the amino acids analyzer system.

The Shimadzu LC amino acids system was used for amino acids detection, equipped with the Shim-Pack Amino-Na column with the following specification (4.6 mm, I.D × 100 mm) containing strong acidic cation exchanger resin (styrene divinyl benzene copolymer with sulphonic groups). The amino acid analyzer is equipped with the auto-injector SIL-10 ADVP for sample injection, SCL-10A VP as system controller, and DGU-14A as degasser. The system flow rate was adjusted to 2 ml/min by using a peristaltic pump (PRR-2A) with column oven CTO-10AV VP set at 60 °C for reaction solutions. The fluorescence detector RF-10A XL was kept at Ex = 350 nm, Em = 450 nm. An ammonia trap column was also used before column elution (Shim-pack ISC30/SO504 Na).

The mobile phases A, B, and C consisted of 0.2 N sodium citrate (pH 3.2), 0.6 N sodium citrate and 0.2 M boric acid (pH 10), and 0.2 M NaOH, marked as MA, MB, and MC, respectively. These mobile phases were run in a 72-min gradient program with 100% MA for 59–72 min; then MB 0%−100% for 14–53 min, and lastly MC 100% for 53.01–58 min.

Pepsin digestibility is determined by the AOAC 971.09 method. Briefly, 1 g of the sample was subjected to 16 h digestion at 42 °C with a warmed solution of pepsin under constant agitation. The insoluble residue was separated by filtration and analyzed for protein by the same method mentioned in AOAC 960.52. The same procedure was applied to all samples to determine the % protein digestibility in all the samples.

Approximately 5–10 g of homogenized sample was taken in a distillation tube, then 2.0 g magnesium oxide and 150 ml distilled water were mixed and subjected to distillation for 5 min on the automatic Kjeldahl Nitrogen Analyzer. In a 250 ml conical flask, 25 ml of 2% boric acid solution was taken, and a few drops of methyl red were added to collect the distillate, then titrated against 0.1 N HCl until a light pink color appeared. The TVBN value was determined by using the following formula (Goulas and Kontominas, 2005).

The O'Fallon et al. (2007) method was followed for fatty acids derivatization. Briefly, 50 μl of extracted oil (fat) was taken in a Pyrex tube, followed by the addition of 1 ml internal standard, 0.7 ml of KOH solution, and about 5.3 ml methanol, capped and mixed thoroughly, and subjected to incubation in a warm water bath at 55 °C for 1.5 h. Then allowed to cool and added 24 N 0.58 ml sulphuric acid and placed in a water bath at 55 °C for 1.5 h. Then allowed the tubes to cool and added 3 ml of hexane, and mixed vigorously on a vortex mixer for 5 min. The fatty acid methyl esters (FAMEs) were extracted in the upper layer (hexane layer), collected, filtered through a 0.45 μ nylon syringe filter, and then analyzed on the instrument.

The prepared samples were analyzed through Gas Chromatography with flame ionization detector (FID) technique with the following instrument specifications: GC-2010, Shimadzu corporation 07947 equipped with FID detector, split injector, and SP-2560 silica fused capillary column (100 m, 0.25 mm, 0.2 μm; Supelco). The instrument operating system includes: injection volume 1 μl with temperature 250 °C, detector temperature 260 °C, column temperature 140 °C for 5 min and then ramped to 240 °C with 4 °C/min, remain stable for 15 min; helium was used as carrier gas with flow rate of 1.12 ml/min and linear velocity of 20 cm/s; split ratio 1:100. Results were expressed as FID response area relative percentages. The results were given as mean standard deviation.

The mineral contents, including sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn), were analyzed by the AOAC methods 999.11 and 985.35. Sample mass (0.5–4.0 g) was adjusted according to the expected mineral concentration of each sample to ensure that analyte levels remained within the linear working range of the instruments. The samples were prepared by accurately weighing 0.5–4 g samples in triplicate in a 100 ml conical flask with the addition of 20 ml of di-acid solution (HNO₃:HClO₄ = 2:1) and kept overnight. The samples were heated on a sand bath at 140 °C for 2 h and H₂O₂ was added until a complete colorless solution was obtained. After cooling, the digested samples were filtered through Whatman filter paper (42) into a 50 ml volumetric flask and made up to the final volume with distilled water. Blank samples were also prepared by following the same procedure. Afterwards, the subsequent determination of elements was performed by electrothermal atomic absorption spectrometry ETAAS/FAAS spectrometry (AOAC, 2023).

A certified reference material from NIST CRM-1515 of dried apple leaves was also prepared in the same way, containing iron, copper, zinc, manganese, sodium, potassium, calcium, magnesium, and other trace elements. Zn, Cu, and Iron were analyzed by Hitachi Z-8000 Atomic Absorption Spectrophotometer with Flame AAS (FAAS) technique with air acetylene. Working standards (0.5–2) mg/L were prepared from ICP grade standards (1,000 mg/L) purchased from Merck (Germany).

The antioxidant activities for each sample were determined by DPPH radical scavenging assay (Karaulova et al., 2021) with some modifications. 1 g of each sample (dried) was dissolved in 10 ml (99.9%) methanol, stirred, and then centrifuged at 3,000 g for 30 min. The supernatant was collected and stored at 4 °C until analysis. For standard preparation, 10 mg of Ascorbic acid was dissolved in 100 ml of distilled water to prepare a 100-ppm solution. Then 20, 40, 60, 80, and 100 ppm concentrations were made.

A 10 μM DPPH solution was prepared by dissolving 0.00394 g of DPPH in 100 ml of methanol. Equal volume (1:1) of the sample extract and DPPH solution was mixed and placed in the dark for 30 min. Absorbance was measured at 517 nm, while methanol was taken as a blank. The experiment was repeated three times at each concentration for all sample solutions.

where “A_control” is the absorbance of the reference solution (methanol + DPPH), “A_sample” is the absorbance of the test solution.

The data collected were analyzed and presented as Mean ± SEM. Statistical analysis was conducted using version 23 of IBM Statistical Package for Social Sciences (SPSS). The data were subjected to a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test, to determine the differences among the groups. Values with p < 0.05 were considered statistically significant. Principal component Analysis (PCA) was performed using XLSTAT (Version 2014.5.03) based on the Pearson correlation matrix, which standardizes variables to account for differences in measurement scales and emphasizes correlations among variables. A distance biplot was generated to simultaneously visualize both samples and variables in reduced-dimensional space, allowing clearer interpretation of patterns, similarities, and differences among the studied species.

The biometric measures of the studied shellfish specimens are listed in Table 1. The examined species showed more than 40% edibility, with values ranging from 28.28 to 72.26%, except for the lobster species. In contrast, condition index values ranged from 29.78 to 75.91%, being highest in cephalopods and lowest in lobsters, with significant variations.

The percentage edibility and condition index values observed among the examined species were generally high, indicating good meat quality. Significant differences (p ≤ 0.05) were found across species, with the highest values recorded in cuttlefish and the lowest in slipper lobster (Table 1). These indices are commonly used in international seafood trade to assess the physiological condition, reproductive phase, and nutritional status of commercial shellfish (Aníbal et al., 2011; Gabbot, 1975; Prato et al., 2019). Previous research has shown that factors such as temperature, feeding, and maturation cycles influence PE and CI values (Okumus and Stirling, 1998; Orban et al., 2006). Seasonal gametogenesis leads to increased nutrient accumulation, while spawning reduces nutrient levels due to energy expenditure (Pillay and Nair, 1973).

A high moisture content is generally considered an indicator of freshness and quality. Moisture was found to be the main component (up to 83%) in the edible portions of the shellfish species under study, with values ranging from 67.1 to 83.5%. Squids and slipper lobsters recorded the highest moisture values (83.5 and 81.8%, respectively). Shellfish are highly perishable, primarily due to their high moisture content, which is influenced by osmotic pressure and environmental conditions (Davies and Jamabo, 2016). Moisture content also determines shelf life and storage conditions (Prato et al., 2019; De Souza et al., 2021). Differences in flavor, texture, and seasonal feeding behavior can further affect moisture levels (Vigneshwari and Gokula, 2018; Umer et al., 2021). The values observed in the present study were consistent with those reported for similar species in prior research (Barrento et al., 2009; Kampouris et al., 2021; Mehta and Nayak, 2017; Sriket et al., 2007; Wen et al., 2014).

Ash values ranged from 0.77 to 1.78% (Table 2) and showed significant variation (p ≤ 0.05) among the species. These differences may result from species-specific traits and habitat differences. The fat content was low across all species (up to 3.1%) but showed significant interspecies variation. Ash and fat content in shellfish showed slight variations, attributed to species differences and habitats. Our findings align with previous studies (Krzynowek, 1987; Kampouris et al., 2021; Laly and Sankar, 2023).

Significant differences (p ≤ 0.05) were observed among the six species of crustaceans and cephalopods regarding protein content, which ranged from 13.43 to 20.27%. Among crustaceans, black tiger shrimp showed the highest protein content (19.62%), followed by cat tiger shrimp, spiny lobster, and slipper lobster. In cephalopods, cuttlefish had the highest protein content (20.27%) compared to squids (15.82%). Protein content in cuttlefish and squids was significantly higher compared to earlier reports (Mehta and Nayak, 2017; Wen et al., 2014). Variations in protein levels across species can be attributed to environmental and biological factors (EFSA, 2014; Venugopal and Gopakumar, 2017), although such factors were not evaluated in this study.

Nutritional energy values were highest in both shrimp species and squids (93.98, 92.52, 91.18 kcal/100 g), while slipper lobster showed the lowest (63.08 kcal/100 g). Higher energy values observed in shrimps and squids are linked to their high protein and low moisture content. Lobsters, having lower protein content, exhibited lower energy yields, consistent with earlier studies (De Souza et al., 2021; Umer et al., 2021). The protein and energy content of shellfish confirm their role as nutritious, low-fat food sources.

The analyzed samples qualify as complete protein sources. Table 3 presents the 18 essential and non-essential amino acids detected in varying quantities. Total amino acids (TAA) were highest in cuttlefish (21.41 g/100 g) and black tiger shrimp (20.65 g/100 g), while the lowest values were recorded in slipper lobster (15.07 g/100 g) and squids (16.92 g/100 g), though not significantly different. Glutamic acid was the most abundant amino acid across all species, followed by aspartic acid, arginine, lysine, and histidine. Essential amino acids lysine (4.23 g/100 g) and histidine (0.40 g/100 g) were significantly higher in cuttlefish and cat tiger shrimp, respectively. Both polar and non-polar amino acids were present in substantial concentrations. Essential amino acids (EAAs) constituted 40%−51% of total amino acids (TAAs) in all species. Total EAAs were highest in cuttlefish (11.00 g/100 g) and black tiger shrimp (9.29 g/100 g). Among non-essential amino acids, values ranged from 8.8 to 11.36 g/100 g, with the highest levels observed in cat tiger shrimp.

The examined species contained essential and non-essential amino acids, including those responsible for flavor enhancement (Fuke and Konosu, 1991; Spurvey et al., 1998). Sweet, umami, and bitter taste components were evident in the amino acid profiles. Flavor amino acids such as glutamic acid, alanine, and glycine were abundant, consistent with other seafood studies (Shao et al., 2014; Temdee et al., 2021; Xu et al., 2022).

The essential amino acid profiles (Table 3) were used to assess protein quality via various nutritional indices: Nutritional Index (NI), Essential Amino Acid Index (EAAI), Predicted Biological Value (p-BV), Predicted Protein Efficiency Ratio (p-PER), and Digestible Indispensable Amino Acid Score (DIAAS).

EAAI was highest in slipper lobster (76.77) and black tiger shrimp (75.63), with a significant difference between the shrimp species. Cuttlefish (61.18) and squids (68.66) showed lower values. EAAI values between 75 and 85 indicate moderate to good-quality protein (Mir et al., 2019; Li et al., 2022). Slipper lobster had the highest EAAI, followed by shrimps. p-BV values ranged from 54.99 to 71.98, with higher values observed in slipper lobster and black tiger shrimp. Black tiger shrimp had the highest NI (10.31–14.84), followed by spiny lobster, cat tiger shrimp, and slipper lobster. In cephalopods, cuttlefish had a higher NI (12.40) than squids (10.86). The biological values indicated efficient nitrogen retention for growth, while NI values were positively correlated with protein content. Differences in EAAI and NI were primarily influenced by essential amino acid composition and genetic or environmental factors (Johansson et al., 2020). The p-PER was also highest in black tiger shrimp and cuttlefish. The p-PER values, though lower than the benchmark of 2.0 for high-quality proteins (Hoffman and Falvo, 2004), were positively associated with leucine and tyrosine content. Comparisons with reported p-PER values in other seafood, such as abalone and scale carp, support this conclusion (Bhat et al., 2022; Shi et al., 2020).

DIAA reference ratios (DIAAR) were generally above 1 for all EAAs, except valine, histidine, leucine, and tryptophan. The DIAAS values were significantly higher in cuttlefish, both shrimp species, and squids, compared to lobsters (Table 4). DIAAS values also confirmed the high quality of shellfish protein. Ratios >1 indicate sufficient digestible indispensable amino acid content, while scores between 75 and 80% for most species signify excellent digestibility and suitability for supplementation (FAO, 2013; Leser, 2013). These results are comparable to prior findings (Shaheen et al., 2016; De Cock et al., 2023). EAAs such as valine, leucine, tryptophan, and histidine, which contribute to various metabolic and immune functions (Holeček, 2018; Zhang et al., 2017), appeared to be the first limiting amino acids in the examined species.

TVBN is a key indicator of seafood freshness. Both shrimp species and cephalopods showed low TVBN values (< 30 mg/100 g) with significant differences between cuttlefish and squid samples (Table 5). All samples were considered fresh and within acceptable limits, except slipper lobster, which had a slightly elevated value (33.35 mg/100 g). TVBN values, an indicator of freshness, remained within acceptable international limits (Ding and Li, 2024; Altissimi et al., 2018). Lower TVBN values in shrimps, squids, and cuttlefish indicate excellent quality. Slightly elevated values in slipper lobsters were still within safe consumption thresholds.

The highest in-vitro pepsin digestibility (>99%) was found in cuttlefish, indicating minimal indigestible protein residue, followed by lobster species and squids. Cat tiger shrimp (96.73%) had significantly lower digestibility than black tiger shrimp (98.56%) and the other species. In vitro pepsin digestibility exceeded 99%, indicating excellent protein absorption potential across species. These values align with previous findings in shellfish and bivalves (Shi et al., 2020; Chasquibol et al., 2023).

Fatty acid profiles revealed high levels of palmitic and oleic acid. Table 6 shows that total saturated fatty acids (SFAs) were highest in cuttlefish (43.42%) and cat tiger shrimp (32.36%), primarily due to palmitic acid (C16:0). Stearic acid (C14:0) was more abundant in squids (23.61%) and cuttlefish (18.98%). Monounsaturated fatty acids (MUFAs) ranged from 17.63 to 48.71%. Oleic acid (C18:1n9c) was highest in cat tiger shrimp (45.29%), followed by slipper lobster (37.6%). Nervonic acid (C24:1n9) was detected in a few species: spiny lobster (0.03%), cuttlefish (0.82%), and squid (0.79%). As a very long chain monounsaturated fatty acid, nervonic acid is recognized for its role in brain development and neurological function, highlighting the potential nutritional relevance of these species (Li et al., 2019).

Polyunsaturated fatty acids (PUFAs) ranged from 15.71 to 28.94%, comprising up to 64.8% of total fat in cat tiger shrimp, which was lower than values reported earlier (Reksten et al., 2024). Omega-3 fatty acids (6.9%−16.21%) and omega-6 fatty acids (2.92%−15.83%) were present in edible tissues of all species, with significant amounts of omega-3 (EPA), although DHA was absent. This is consistent with previous reports that DHA is typically localized in fish brain and retina tissues (Sun et al., 2018; Rincón-Cervera et al., 2020). Comparatively, the shrimps, lobsters, squids, and cuttlefish contain an excellent amount of EPA, which exceeds the recommended dietary intake of EPA + DHA (250 mg or 0.25 g/day) suggested by the FAO [Codex Alimentarius Commission (CAC), 2016].

The omega-3/omega-6 ratio was greater than 1 in most species except cat, tiger shrimp, and slipper lobster, indicating their potential role in reducing inflammation and supporting cardiovascular health. Among all species, the cat tiger shrimp have the highest PUFA/SFA ratio (0.62) compared to the recommended lowest value (0.45) established by Department of Health and Social Security (DHSS) (1994); higher PUFA/SFA ratios significantly lower the risk of coronary heart disease (Chakma et al., 2024). The fat nutritional health indices, AI and TI of the examined species were within the recommended value < 1 suggested by Fernandes et al. (2014); except for cuttle fish that showed slightly higher values (1.97 AI; 1.08 TI), which could be harmful to human health (Sumi et al., 2025). In the current investigations, the H/H (1.15–2.14), HPI (1.13–2.00), and FLQ indices (0.04–2.82) values exceeded 1 except few species (Table 6). These values aligned with the previous findings, while considering suitable for human consumption and wellbeing (Chen and Liu, 2020; Sumi et al., 2025).

Mineral content in edible muscles of six shellfish species is depicted in Table 7. The macro mineral phosphorus (P) was detected in highest concentration (85–135 mg/100 g) followed by magnesium (19.11–42.26 mg/100 g) and potassium (1.41–16.34) while lower concentrations of calcium (1.09–16.02 mg/100 g) and sodium (0.12–0.42 mg/100 g) were detected in all examines samples. In case of trace minerals, higher levels of iron (0.11–0.88 mg/100 g) and zinc (0.22–0.77 mg/100 g) than manganese (0.03–0.11 mg/100 g) were recorded. Comparatively highest concentration of phosphorus was detected in spiny lobster and cuttle fish; magnesium was highest in slipper lobster and Indian squids, whereas trace elements iron and zinc were predominant in cat tiger shrimp and cuttle fish, respectively.

The resultant data on the mineral composition of examined species displayed compelling differences with the earlier reports (Bhatti et al., 2025; Sreelakshmy et al., 2024), associated with dietary pattern and bioavailability of minerals in the surrounding environment (Rodrigues et al., 2021). The shrimp byproducts are rich in calcium and magnesium (Liu et al., 2021); thus, lower calcium levels in edible muscles of examined species aligned with the fact that calcium is the major element of the skeletal system and exoskeleton in vertebrates and crustaceans, followed by phosphorus and magnesium (Truong et al., 2023; Coelho et al., 2024).

The shellfish methanolic extracts showed strong antioxidant activity against the free radical DPPH, which ranged from 47.63 to 68.10% summarized in Table 8. Spiny lobster exhibited the highest antioxidant activity, significantly higher than the shrimp species. The lowest RSA was found in cat tiger shrimp (47.63%), while both squid and cuttlefish showed >50% activity. Since methanol predominantly extracts low-molecular-weight polar secondary metabolites, particularly phenolic compounds, the observed antioxidant activity is more likely attributable to methanol-soluble phenolics and metabolites (Ignat et al., 2011). Although bioactive peptides and free amino acids exhibit antioxidant properties, their extraction into pure methanol is limited, and thus their direct contribution under the present extraction conditions cannot be confirmed (Sila and Bougatef, 2016). Future studies employing aqueous or hydrolyzed extracts, along with targeted peptide-fraction analyses, are required to verify their specific roles in antioxidant capacity.

Earlier studies confirmed the use of marine-based products as natural antioxidants to prevent food spoilage (Boztas et al., 2019), while various extractions of crustaceans and seafood by-products, Marine-Based Foods (MBFs), shrimp paste, and amino acids, have been shown to neutralize DPPH radical scavenging activity. Accordingly, the present study findings are consistent and comparable with the previous in-vitro studies (Olatunde et al., 2019; Xu et al., 2017; Prapasuwannakul and Suwannahong, 2015; Kurniawan et al., 2020).

PCA was employed to integrate proximate composition, fatty acid and amino acid profiles, and nutritional indices of the studied shellfish species. The scree plot (Figure 1) showed a clear inflection indicating that the first two principal components adequately explained the dataset. PC1 and PC2 together accounted for 79.84% of the total variance, with PC1 explaining 48.56% and PC2 accounting for 31.28%, confirming the robustness of the two-dimensional PCA model (Table 9).

The PCA biplot (Figure 2) revealed distinct separation of species based on their nutritional characteristics. S. pharaonis and P. monodon were positioned on the positive side of PC1, closely associated with protein content, total amino acids (ΣAAs), essential amino acids (ΣEAAs), saturated fatty acids (ΣSFAs), and n-3 fatty acids, indicating a protein and amino acid-rich nutritional profile. In contrast, P. pulchricaudatus and T. indicus clustered on the negative side of PC1, which was associated with total fat, monounsaturated fatty acids (ΣMUFAs), polyunsaturated fatty acids (ΣPUFAs), and n-6 and n-9 fatty acids, reflecting lipid-dense profiles dominated by unsaturated fatty acids. PC2 was mainly influenced by moisture content, with opposite contributions from ash and energy value, suggesting an inverse relationship between water content and nutrient density. P. ornatus and U. duvaucelii occupied intermediate positions, indicating balanced contributions from both protein and fat-related variables.

Overall, the combined interpretation of the scree plot and PCA biplot confirms that protein quality and fatty acid composition are the principal factors governing nutritional differentiation among the studied species, highlighting their potential for targeted dietary and functional food applications.