Bioactive Ingredients, Functions, and Development Strategies of Phascolosoma esculenta—An Edible Marine Organism: A Review (2025)

2.1 Ferritin

Ferritin is a kind of functional protein widely found in bacteria, plants, and animals, which plays an important role in iron metabolism, both in detoxifying intracellular iron and in storing iron (Arosio etal.2009). Over 50 ferritin crystal structures have been identified, mostly from bacterial ferritins, and few of the nonbacterial ferritins are from marine invertebrates. For example, Chaetopterus ferritin from Chaetopterus sp. (parchment tube worm) was the first crystal of ferritin isolated from the marine invertebrates, but the mechanism by which it stores iron is unknown (De Meulenaere etal.2017). Currently, two species of P. esculenta ferritin have been reported.

Du etal. acquired the gene sequence of P. esculenta ferritin (PeFer) using the RACE method. The sequence is 1017 bp long, comprising a 5′-terminal noncoding region of 151 bp, a 3′-noncoding region of 341 bp, and an open reading frame of 525 bp (including a stop codon), capable of encoding 175 amino acids, and the polyclonal antibody was prepared (Du etal.2008; Su etal.2009). Ding etal. conducted recombinant purification of PeFer in Escherichia coli and observed that under various heavy metal treatments, the recombinant PeFer exhibited distinct conformational changes and formed diverse aggregates, suggesting that PeFer may sequester different heavy metals through unique amino acid binding sites. Additionally, PeFer exhibits potent oxidative catalytic activity towards Fe and enrichment of toxic metal ions (Mn²⁺, Pb²⁺, Cd²⁺, Cr³⁺ and Cu²⁺, etc.) compared to nontoxic metal ions (Sn²⁺ and Zn²⁺) (Ding etal.2018). Fer147, a new protein screened for interaction with PeFer using a yeast two-hybrid system, has a full length of 916 bp, without a signal peptide, and it also belongs to the ferritin superfamily. The invertebrate ferritin contains only one subunit, whereas Fer147 has both the iron oxidase center of the H subunit and the iron nucleation site of the L subunit, indicating that Fer147 is functionally equivalent to both subunits of vertebrate ferritin (Ding etal.2017). PeFer and Fer147(PDB: 6LPD, 6LPE) have similar structures to other known ferritins, but in terms of detailed structure, the triple channel of Fer147 and the quadruple channel of PeFer have different variations in electrostatic potential, and the two differ significantly in aggregating metal ions and excluding cations (Ming, Huan, etal.2021). The enrichment ability of these two ferritins to heavy metals was better than that of natural horse-spleen ferritin, and the enrichment ability of Fer147 to toxic heavy metals such as Cu²⁺, Pb²⁺ and Hg²⁺ was higher than that of PeFer (Ding etal.2017). Through proteomic and metabolomic analyses, Ming etal. discovered that PeFer exerts a protective effect against Cd-induced injury to bone marrow mesenchymal stem cells (BMSCs). It impedes BMSC proliferation by inducing G0/G1 cell cycle arrest and apoptosis, while safeguarding BMSCs from Cd-induced apoptosis via energy metabolism (Ming, Wu, etal.2021).

Iron ferritin is well known for its significant flexibility and adaptability in metal absorption, and it is considered a promising candidate for heavy metal detoxification and environmental detection agents. Furthermore, ferritin possesses a unique structure. A typical ferritin consists of 24 interconnected subunits arranged in the form of four-helix bundles, creating a symmetrical hollow cage-like shell with 4–3–2 symmetry that closely resembles a spherical shape. This shell exhibits excellent stability and is resistant to high temperatures, acids, and alkalis (Zang etal.2017). Through a reduction reaction, the iron core in ferritin can be removed, resulting in the formation of apoferritin shells. Currently, nanotechnology based on ferritin has been developed, allowing the loading of small molecules into this shell-like biological model to serve as carriers for novel bioinorganic nanoparticles (Li etal.2007). As a naturally occurring nanocarrier in the environment, PeFer and Fer147 effectively reduce the quantity of organic solvents used in the preparation process, making it a potential environmentally friendly nanotechnology (Khoshnejad etal.2018). Therefore, P. esculenta ferritin is expected to be developed as a natural nanoparticle carrier, with its strong ability to enrich metal ions and also making it suitable for transporting calcium, iron, and other essential elements required by the human body.

2.2 Trx-Like Protein

Thioredoxin (Trx), a highly conserved antioxidant molecule, constitutes one of the principal antioxidant systems crucial for maintaining intracellular redox homeostasis. Excessive intracellular reactive oxygen species (ROS) can induce cellular oxidative damage or apoptosis. Cd²⁺, either directly or indirectly, can elevate intracellular ROS levels (Lu and Holmgren2014). To elucidate the molecular response of Trx to Cd stress, Meng etal. identified a Trx isoform, named PeTrxl, from P. esculenta, aiming to enhance understanding of the role of Trx-like protein 1 in intracellular ROS regulation under Cd stress. Experimental findings revealed the highest Trx levels in the body fluids of P. esculenta. The expression of PeTrxl mRNA markedly increased after 12 and 24 h of exposure to both low and high Cd concentrations, while the expression of Trx-like protein 1 mRNA significantly decreased after 96 h of exposure. This suggests that Trx-like protein 1 mRNA is responsive to Cd stress at early stages. The concentration of ROS in the coelomic fluid exhibited a notable increase subsequent to the administration of PeTrxl into P. esculenta. Insulin disulfide reduction and 2′2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging experiments suggested that PeTrxl may exert its antioxidant function by reducing disulfide bonds in proteins or directly scavenging ROS (Meng etal.2021). Gu etal. studied the PeTrx2 gene from the mitochondria of P. esculenta. Under cadmium stress, PeTrx2 in the gut of P. esculenta exhibited a rapid response to oxidative stress. Invitro, recombinant rPeTrx2 demonstrated dose-dependent REDOX activity, ABTS free radical scavenging ability, and enhanced cadmium tolerance in E. coli. After RNA interference with PeTrx2, proapoptotic genes (Caspase-3 and Bax) were significantly upregulated, while antiapoptotic genes (Bcl-2 and Bcl-XL) were downregulated, confirming that PeTrx2 mitigates cadmium toxicity by regulating mitochondria-dependent apoptotic pathways (Gu, Zheng, etal.2024). Trx-like protein 1 could serve as a biomarker for investigating the detoxification mechanism of invertebrate mudflat organisms exposed to heavy metals.

2.3 Hemerythrin

Hemerythrin serves as an oxygen carrier and was initially identified in marine invertebrates. In contrast to hemoglobin, hemerythrin utilizes a nonheme diiron active site, mitigating many stress-induced side reactions associated with hemoglobin, including autoxidation and nitric oxide reaction (Fischer-Fodor etal.2011). Wang etal. generated a cDNA library of P. esculenta with a capacity of 3.06 × 105 cfu. The full-length cDNA sequence of hemerythrin comprised 823 bp, encoding 120 amino acids. The protein had a molecular weight of 13.63 kDa and an isoelectric point of 5.78 (Wang, Li, etal.2010; Wang, Su, etal.2010). The sequencing of hemerythrin in P. esculenta serves as a valuable addition to hemerythrin research, facilitating further investigation into its potential as a raw material for artificial oxygen carriers, such as “blood substitutes.”

2.4 ACE Inhibitory Peptides

Hypertension is a prevalent clinical condition with an escalating incidence over time. The renin–angiotensin system, particularly the angiotensin I-converting enzyme (ACE), plays a pivotal role in blood pressure regulation (Daskaya-Dikmen etal.2017). Synthetic ACE inhibitors have been developed, such as benazepril, enalapril, and perindopril, which have been shown to have good antihypertensive effects (Piepho2000). However, these synthetic substances are prone to cause adverse side effects. There has been a trend to develop antihypertensive peptides from natural foods.

Du etal. isolated an ACE inhibitory peptide from P. esculenta with a molecular weight of 1222.7 Da and an amino acid sequence Ala-Trp-Leu-His-Pro-Gly-Ala-Pro-Lys-Val-Phe, suggesting that its ACE inhibitory activity may stem from the Phe residue at the C-terminal and Ala residue at the N-terminal, flanked by two positively charged amino acids, His and Lys (Du etal.2013).

Wu etal. utilized BIOPEP to screen 22 proteins of P. esculenta and employed the LibDock module in Discovery Studio 3.5 software for molecular docking experiments to assess the inhibitory potential of the screened peptides on ACE. Ninety-nine ACE inhibitory peptides exhibited IC₅₀ values below 50 μM, and nine peptides were synthesized for validation. The IC₅₀ values ranged from 3.43 to 4.38 μM, with an error margin of less than 1.0 unit (Wu, Liu, etal.2014; Wu, Fang, etal.2014).

Guo etal. isolated three ACE inhibitory peptides, RYDF (IC₅₀ = 235 μM), YASGR (IC₅₀ = 184 μM), and GNGSGYVSR (IC₅₀ = 29 μM), with molecular weights of 600, 553, and 896, respectively, through pepsin–trypsin hydrolysis of water-soluble proteins from P. esculenta. Automated molecular docking was employed to investigate the interaction between ACE and these peptides. GNGSGYVSR exhibited the highest ACE inhibitory activity and the lowest Ki value. It interacted with 7 hydrophobic residues and 12 hydrophilic residues of ACE, including Glu384, a critical residue for ACE-Zn²⁺ binding. This peptide may enhance ACE inhibition by disrupting ACE-Zn²⁺ binding. Compared to the other two peptides, GNGSGYVSR exhibited stronger and more stable residue binding, significantly reducing systolic blood pressure in spontaneously hypertensive rats from 228 mmHg to 197 mmHg at 2 h (p < 0.05) and maintaining this effect for 4 h. Thus, GNGSGYVSR emerges as a promising candidate for blood pressure regulation (Guo etal.2017).

2.5 Fibrinolytic Enzyme

Fibrinolytic enzymes dissolve fibrin clots and are commonly employed as drugs to prevent thrombosis. Their advantage over anticoagulants and antiplatelet agents lies in their ability to directly target existing thrombi (Labrou2019).

Current research on P. esculenta fibrinolytic enzyme focuses primarily on extraction and separation processes. Cai etal. employed homogenization, extraction, centrifugation, Sephadex G-25 desalting, and DEAE Sepharose Fast Flow gradient elution to isolate P. esculenta fibrinolytic enzyme, which has a relative molecular mass of 32 Ku. P. esculenta fibrinolytic enzyme primarily resides in the intestinal tract. It exhibits some thermal stability but is sensitive to high temperatures, displaying optimal stability at 37°C and pH 6–9. Compared to the positive control urokinase, P. esculenta fibrinolytic enzyme exhibits direct fibrin degradation and thrombolytic effects (Cai, Zhou, etal.2021; Cai, Xing, etal.2021). P. esculenta fibrinolytic enzyme holds promise for the development of novel thrombolytic drugs.

2.6 Superoxide Dismutase

Superoxide dismutase (SOD) plays a crucial role in neutralizing superoxide anion radicals generated by extracellular stimuli and by-products of oxygen metabolism from certain mitochondrial substrates (McCord and Fridovich1969). Three isoforms of SOD have been identified: copper–zinc SOD (Cu/ZnSOD), manganese SOD (MnSOD), and extracellular SOD (Parge etal.1992). P. esculenta possesses 12 copies of the Cu/ZnSOD gene and 15 copies of the MnSOD gene, the highest number of copies reported in the published annelid genome (Zhong etal.2022).

Wang etal. isolated a complete MnSOD cDNA from P. esculenta. The cDNA spans 1385 bp, comprising an open reading frame of 681 bp that encodes 226 amino acids. The predicted protein has a molecular weight of 25.2 kDa and a theoretical isoelectric point of 5.96 (Wang, Li, etal.2010; Wang, Su, etal.2010). The Cu/ZnSOD cDNA, cloned by the Liu group, spans 857 bp, comprising a 75 bp 5' UTR, a 323 bp 3' UTR, and a 459 bp open reading frame, encoding 152 amino acids. The predicted molecular weight of the protein is approximately 15.6 kDa, with a theoretical isoelectric point of 5.65. The recombinant MnSOD from P. esculenta, expressed in E. coli, exhibited enhanced expression following exposure to heavy metal and temperature stress. Recombinant Cu/ZnSOD increases metal tolerance of E. coli under Cd stress, and shows antioxidant activity and scavenging ability of free radicals (Liu etal.2022). Therefore, P. esculenta SODs can be used as indicators for the evaluation of heavy metals or developed as antioxidant drugs.

2.7 Polysaccharides

Aquatic organisms typically contain higher levels of polysaccharides compared to terrestrial counterparts. These polysaccharides exhibit anti-inflammatory (Miller etal.1993), anticoagulant (Mourão etal.1996), antitumor (Hsu etal.2018), and antioxidant effects (Li etal.2015), rendering them valuable for development (Xiong etal.2020).

Liang etal. determined that the optimal conditions for extracting P. esculenta polysaccharides were at 40°C extraction temperature, a phosphate buffer to raw material ratio of 2:1, an extraction time of 5.5 h, and a trypsin to raw material ratio of 1.6:1, resulting in the highest yield (Liang2008). Wu etal. prepared P. esculenta polysaccharides by enzymatic digestion. The resulting P. esculenta polysaccharides exhibited a monosaccharide composition comprising mannose, ribose, rhamnose, glucuronide, glucose, galactose, xylose, arabinose, and caramel, with mass ratios approximately 3:2:1:1.6:7.6:5.5:1.5:1:3 (Wu etal.2020). Additionally, P. esculenta polysaccharides exhibit notable invitro free radical scavenging activity and can substantially impact lipid metabolism-related indicators in hyperlipidemic mice, resulting in a significant hypolipidemic effect. PEP-1 exhibited higher antioxidant capacity compared to PEP-2, and it also enhanced the oxidative stress tolerance ability of P. esculenta. Furthermore, experiments revealed that mice with high-fat-induced liver conditions exhibited substantial fatty changes and cystic degeneration. However, these histopathological alterations were notably mitigated following treatment with P. esculenta polysaccharides, indicating a potential improvement in histological alterations. Zhou etal. isolated two polysaccharides, PEP-1 and PEP-2, from P. esculenta using column chromatography. These polysaccharides have molecular weights of 33.6 kDa and 5.7 × 10³ kDa, respectively. Their antioxidant capacities were evaluated in Caenorhabditis elegans, revealing that PEP-1 exhibits superior efficacy compared to PEP-2. Furthermore, PEP-1 significantly enhances the oxidative stress tolerance of C. elegans (Zhou etal.2024).

2.8 Oligosaccharides

Oligosaccharides, formed by the dehydration and condensation of 2–10 monosaccharide molecules or through the degradation of polysaccharides, exhibit high polarity and low degree of polymerization, often accompanied by specific pharmacological activities. Oligosaccharides have the characteristics of high polarity and low degree of polymerization, and often have certain pharmacological activities. Human milk oligosaccharides, for instance, promote infant intestinal and immune development (Donovan and Comstock2016).

Yang etal. conducted the first preparation of P. esculenta oligosaccharides via enzymatic digestion. They characterized these oligosaccharides using liquid chromatography-mass spectrometry and Fourier-transform infrared, and established a mouse model of E. coli-induced sepsis to examine the protective effects of P. esculenta oligosaccharides. Analysis revealed that P. esculenta oligosaccharides primarily consist of D-glucosyl and D-galactosyl, with smaller amounts of D-mannosyl and D-arabinosyl. Sharp absorption peaks at 841.5 cm⁻¹ and 892.5 cm⁻¹ in the Fourier-transform infrared spectra suggest the presence of both α-type and β-type glycosidic linkages in P. esculenta oligosaccharides. Mice with E. coli-induced sepsis, treated with P. esculenta oligosaccharides at all three doses, exhibited significantly increased survival rates and notable suppression of bacterial burden in the blood and liver. These results suggest that P. esculenta oligosaccharides may be enhancing survival through systemic bacterial clearance. Furthermore, administration of all three doses of P. esculenta oligosaccharides led to elevated levels of the anti-inflammatory cytokine interleukin-10 (IL-10), accompanied by significant reductions in the secretion of pro-inflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in mice receiving doses of 10 and 50 mg/kg. These findings indicate the potential of P. esculenta oligosaccharides to mitigate systemic and organ inflammation induced by E. coli infection through upregulation of IL-10 expression, thereby reducing TNF-α and IL-1β-related damage (Yang etal.2019).

2.9 Summary Section

As above, the reported primary bioactive constituents of P. esculenta comprise polysaccharides, oligosaccharides, peptides, and functional proteins, as illustrated in Table2. It is important to note that the current research on the composition of P. esculenta is far from sufficient. The discovery of new drugs and drug candidates from marine organisms is very promising, and many drugs based on marine organisms have been developed (Table1). In the future, we can excavate more active compounds, including small molecule compounds, from the P. esculenta.

3.1 Antibacterial Activity

The coelomic fluid of P. esculenta harbors most of its antimicrobial active substances, whereas the body wall is typically consumed as food, leading to the underutilization of the coelomic fluid. Extracting antimicrobial agents from this fluid can optimize the utilization of P. esculenta, minimizing resource wastage and environmental pollution. Liang etal. employed three methods of direct centrifugation, ultrasonic centrifugation, and 2% acetic acid extraction to treat the body cavity fluid of P. esculenta, respectively. Among these, the supernatant obtained through ultrasonic centrifugation exhibited the most pronounced inhibitory effect on Gram-positive bacteria, while its effects on Gram-negative bacteria and fungi were less significant. The supernatant obtained by the other two methods showed no significant antibacterial activity (Liang etal.2015). Meanwhile, Zhang etal. observed that P. esculenta coelomic fluid inhibited E. coli, Shewanella septicum, and Vibrio parahaemolyticus but had negligible effects on Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Micrococcus lysodeikticus. Isolation and purification from P. esculenta coelomic fluid yielded a 10–15 kDa small molecule active protein with antibacterial properties, likely constituting one of the antibacterial components in the organism (Zhang etal.2020). Additionally, Shu etal. (Shu etal.2023) isolated an antimicrobial peptide from the leukocytes of P. esculenta coelomic fluid, exhibiting efficacy against Gram-positive bacteria (S. aureus, Bacillus cereus) and Gram-negative bacteria (E. coli, Vibrio harveyi), along with robust resilience to high temperatures and alkalinity, and displaying nonhemolytic and protease resistance.

3.2 Cardio-Cerebrovascular Protection

Wu etal. established a hyperlipidemia mouse model and observed that P. esculenta polysaccharides reduced total cholesterol, triglycerides, low-density lipoprotein cholesterol, and the atherosclerosis index in the serum, indicating potential hypolipidemic effects. Moreover, P. esculenta polysaccharides increased serum SOD activity and reduced malondialdehyde (MDA) levels, thereby lowering serum free radicals and enhancing antioxidant capacity. Consequently, it is speculated that P. esculenta polysaccharides’ hypolipidemic function may be mediated through the antioxidant pathway (Wu etal.2020).

3.3 Liver Protection

Phascolosoma esculenta polysaccharides significantly reduce liver coefficient, as well as levels of aspartate aminotransferase and alanine aminotransferase in liver tissue, promoting liver cell repair (Wu etal.2020). Wu etal. conducted experiments using pepsin alone or in sequence with pepsin-trypsin to digest water-soluble and insoluble proteins from P. esculenta. They discovered that proteins digested with pepsin-trypsin exhibited higher ACE inhibitory activity invitro, with hydrolysates of insoluble proteins demonstrating greater ACE inhibitory activity than soluble ones. Invivo experiments on spontaneously hypertensive rats showed that pepsin-trypsin hydrolysates significantly reduced both systolic and diastolic blood pressure, confirming their efficacy (Wu, Liu, etal.2014; Wu, Fang, etal.2014). Additionally, three peptides—RYDF, YASGR, and GNGSGYVSR—derived from P. esculenta also lowered systolic and diastolic blood pressure in spontaneously hypertensive rats, with GNGSGYVSR exhibiting the most pronounced effect, followed by YASGR, and RYDF showing a lesser effect (Guo etal.2017). Cai etal. conducted invitro experiments to assess the dissolution effect of fibrinolytic enzyme from P. esculenta on blood clots, revealing an 88.09% dissolution rate after 3 h (Cai, Zhou, etal.2021; Cai, Xing, etal.2021).

3.4 Antioxidant Activities

Chen etal. isolated proteins from five components of the luminal fluid of P. esculenta, focusing on the two proteins, PEP II and PEP V. The IC₅₀ values of PEP II and PEP V for hydroxyl radicals were 1.637 and 0.998 mg/mL, respectively, while the IC₅₀ values for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were 5.581 and 2.801 mg/mL, respectively. The IC₅₀ values for total reducing power were 3.700 and 1.461 mg/mL, respectively. Based on these three indices, both proteins exhibited significant antioxidant activity, though they showed considerable differences in DPPH radical clearance and total reducing power. Protein secondary structure analysis revealed that the secondary structure of PEP II consisted of β-sheet (40.29%) > β-turn (36.21%) > α-helix (23.50%) > random (0%), while PEP V's secondary structure was composed of β-turn (38.28%) > α-helix (21.74%) > random (20.54%) > β-sheet (19.44%). The authors further analyzed the relationship between secondary structure and antioxidant activity, speculating that β-sheet may be associated with hydroxyl radical scavenging, while random coils and β-turns appear to be positively correlated with DPPH radical clearance and total reducing power (Chen etal.2021). Many compounds in P. esculenta display antioxidant capacities, such as P. esculenta polysaccharide, which exhibits significant scavenging activity for DPPH and superoxide anion radicals, potentially contributing to its hypolipidemic and hepatoprotective effects through antioxidant mechanisms (Wu etal.2020). Oxidative stress is a crucial mechanism in sepsis pathogenesis. P. esculenta oligosaccharides enhanced glutathione peroxidase (GSH-Px) and SOD enzyme activities while significantly inhibiting MDA activity in administered mice, demonstrating substantial antioxidant capacity and the ability to ameliorate oxidative stress. Both peptides and ferrous chelating-peptides extracted from P. esculenta significantly enhanced the spatial learning memory of rats. Moreover, ferrous chelating-peptides exhibited superior enhancement compared to peptides alone, indicating that chelation enhances peptide function. The impact of peptides and ferrous chelating-peptides on spatial learning memory in mice may be attributed to peptides with a higher hydrophobic ratio affecting the invivo antioxidant capacity of ferrous chelating-peptides by stabilizing ROS (Yang etal.2019).

3.5 Other Activities

Phascolosoma esculenta exhibits diverse activities including antifatigue effects, enhanced learning and memory, and immune regulation. Feeding mice with P. esculenta significantly increased liver glycogen and muscle glycogen content, resulting in prolonged swimming times (Niu and Tang2012). Peptides and ferrous chelating-peptides derived from P. esculenta significantly enhanced spatial learning and memory in rats, with ferrous chelating-peptides showing superior efficacy to peptides, indicating the enhancing effect of chelation on peptides function. The improved spatial learning and memory associated with P. esculenta peptides and ferrous chelating-peptides may be attributed to their high amino acid content and their ability to up-regulate the expression of NR2A and NR2B, which are receptors related to learning and memory. Moreover, P. esculenta peptides can increase the expression of genes related to oxidative stress, thereby enhancing learning and memory. Additionally, P. esculenta peptides can enhance learning and memory by up-regulating genes associated with oxidative stress (Liu etal.2016). P. esculenta polysaccharides have been shown to increase the indices of liver, spleen, and thymus in mice, indicating its immune-regulating function similar to many marine organisms (Liang2008).

4.1 Adaptability to Heavy Metals

Heavy metal pollution is a prevalent environmental issue in marine ecosystems. Due to economic development and human activities, metals such as Cu, Pb, Ni, Cd, Zn, Fe, and Hg, released by industrial processes, have caused significant harm to both the environment and marine organisms (Traina etal.2019). High concentrations of heavy metals can adversely affect P. esculenta. Gao etal. conducted an analysis of heavy metal content in sediment samples from various marine regions in Guangxi, Fujian, and Zhejiang. They discovered a higher distribution of essential trace elements such as Zn, Cu, Mn, and Fe in the muscle tissue of the species. Higher levels of Zn and Fe within the safe concentration range correspond to greater nutritional value. Additionally, traces of harmful elements such as Pb, As, Cd, and Hg were detected, albeit in small quantities. Among these metals, Cd and Hg exhibited the highest enrichment coefficients (Gao etal.2012). Exposure to heavy metals Cd²⁺ and Hg²⁺ induced alterations in body fat, protein content, and fat content of body wall muscles. Higher metal concentrations resulted in more pronounced growth inhibition, and under the combined stress, the growth of P. esculenta almost stops. Nonetheless, in environments where metal stress exceeds 60 times the national fishery water quality standard, albeit with sluggish growth, mortality did not occur, suggesting robust metal tolerance in P. esculenta (Wu etal.2015). This phenomenon may be associated with ferritin and metallothionein and antioxidant-related mechanisms within the body of P. esculenta. Ferritin functions in heavy metal sequestration and detoxification, while metallothionein can provide protection against acute Cd poisoning. Notably, earthworms, also invertebrates, thrive in Cd-rich soil, underscoring the crucial role of metallothionein. Nonetheless, studies on metallothionein in P. esculenta are lacking. Gu etal. identified a metallothionein (PeMT) and a metal-responsive transcription factor 1 (PeMTF1) in P. esculenta. PeMT exhibited the highest expression in the gut, followed by the coelomic fluid, renal duct, constrictor muscle, and body wall. PeMTF1 was highly expressed in the body cavity fluid, contractile muscle, and intestinal tract. Under Zn stress, PeMT and PeMTF1 expression in the intestine of P. esculenta initially increased and then decreased. They then recombinantly expressed PeMT protein (PGX-6P-1-MT), which significantly enhanced Zn tolerance in E. coli and exhibited dose-dependent ABTS free radical scavenging activity. After RNA interference with PeMT, MDA content, SOD activity, GSH content, and Caspase-3/8/9 activity significantly increased, indicating that PeMT plays a crucial role in chemical resistance and antiapoptotic processes. Furthermore, RNA interference with PeMTF1 led to a significant decrease in PeMT expression (by 53.95%), confirming the regulatory role of PeMTF1 in PeMT expression (Gu, Wang, etal.2024). Furthermore, the accumulation of heavy metals by P. esculenta can serve as an indicator for ecological monitoring, facilitating the assessment of environmental metal pollution severity in coastal ecosystems.

4.2 Adaptability to Temperature

Ocean acidification and warming are poised to significantly impact the survival of planktonic larvae of marine invertebrates. However, marine species possess adaptive mechanisms to contend with environmental changes, including phenotypic plasticity or microgenetics. Particularly noteworthy is the adaptability of species inhabiting intertidal zones to fluctuations in temperature and pH (Foo and Byrne2016). Nonetheless, extremes in temperature, whether too high or too low, can precipitate detrimental effects. Heat shock proteins (HSP) play a crucial role as molecular chaperones, being stress proteins synthesized by organisms in response to stress-induced protein denaturation. Instances such as extreme cold temperatures, excessive cellular energy depletion, toxicity, and other extreme concentrations can trigger the expression of HSP. Invertebrates dwelling in the ocean's tidal zone undergo a substantial increase in body temperature following low tide, leading to the upregulation of HSP expression (Feder and Hofmann1999). Among the various families of HSPs, HSP70 and HSP90 stand out; however, while HSP70 has been subject to extensive study in P. esculenta, research on HSP90 remains limited.

Li etal. conducted the cloning of HSP90 from P. esculenta, revealing a sequence spanning 2521 bp. This sequence encompasses a 5' untranslated region of 110 bp, a 3' untranslated region of 230 bp, and an open reading frame of 2181 bp. Investigation into the expression of P. esculenta HSP90 messenger RNA followed exposure to heavy metals and heat stress. The findings indicate that HSP90, belonging to the HSP90 family, exhibits high conservation levels and functions as an intracellular, non-secretory protein. The upregulation of HSP90 expression occurred to varying degrees in response to the presence of Cd²⁺, Zn²⁺, and Cu²⁺, and elevated temperatures, suggesting its involvement in regulating heavy metal and heat stress in P. esculenta (Li etal.2012). Su etal. similarly cloned the gene encoding HSP70 from P. esculenta. The cDNA sequence comprises 2520 bp, with a 5'-terminal untranslated region of 125 bp, a 3'-terminal untranslated region of 421 bp containing the typical polyadenylate signal sequence AATAAA, a poly(A) tail, and an open reading frame of 1974 bp. The expression of HSP70 in P. esculenta was induced by the presence of Zn²⁺, Cd²⁺, and elevated temperatures (Su etal.2010).

Gao etal. conducted a study on the physiological and tissue changes in P. esculenta under acute heat stress. Following exposure to heat stress, there was a significant increase in the concentration of MDA and the activities of SOD and GSH-Px in the coelomic fluid. Moreover, the expression of HSP70 and HSP90 in both the coelomic fluid and intestine was upregulated. Exposure to 40°C for 96 h resulted in visible cracks in the muscle layer of the tightly bound body wall and the stretched muscle layer, accompanied by alterations in the nuclei of muscle cells (Gao etal.2022). Additionally, another investigation reported damage to the body wall and kidney of P. esculenta following 96 h of stress at 5°C. This stress led to the separation and rupture of the stratum corneum of the body wall and disordered arrangement of the single columnar epithelium in the renal epithelium. Furthermore, enlargement of the lumen formed by the outer membrane of the bottled protrusion and breakage of the circular muscle were observed (Shen etal.2021). The expression levels of both HSP70 and HSP90 in P. esculenta were significantly elevated under conditions of both high and low temperature stress.

4.3 Adaptability to Salinity

Many marine invertebrates have developed adaptations to thrive in fluctuating salinity environments. However, high salinity estuaries exhibit low species richness, and species with high salt tolerance cannot survive below 5 ppt (Henry etal.2012). In response to salinity stress, P. esculenta undergoes physiological adjustments, regulating enzyme activities and immune functions to mitigate damage induced by osmotic pressure fluctuations. The optimal salinity range for P. esculenta growth is 10–35 ppt, with peak activity observed at 25 ppt. Activity nearly ceases in both high and low salinity conditions, with the rhynchodaeum retracting (Zheng etal.2017). Under low salt conditions, the activities of Na⁺/K⁺-ATPase, acid phosphatase, and alkaline phosphatase initially increase and then decline before stabilizing (You etal.2019). Low salt stress can induce renal muscle disorganization, necrosis, and expansion of the cytoplasmic sacs. When salinity is below 10‰, P. esculenta cannot survive. The lowest total SOD activity and highest MDA concentration were observed in the death group, indicating a breakdown of the antioxidant system and enhanced lipid peroxidation due to low salt stress. Transcriptomic analysis of differentially expressed genes suggested that low salt stress may lead to mortality through mechanisms such as disruption of ion transport and immune responses (Hu etal.2024).

4.4 Adaptability to Hypoxic

Invertebrates inhabiting the intertidal zone of the ocean exhibit greater adaptability to anoxic-reoxygenation conditions and demonstrate higher tolerance to hypoxic stress than other organisms (Ivanina etal.2016). Xing etal. investigated the effects of hypoxic stress on the morphology, physiology, and biochemistry of P. esculenta. They found that after 7 days of hypoxia, the body turned black but did not die. Upon reoxygenation, the body became brown and malleable. Total antioxidant capacity (T-AOC) increased, while MDA and lactate dehydrogenase (LDH) levels decreased, indicating that energy supply was maintained by enhancing antioxidant capacity and initiating anaerobic metabolism. Transcriptomic analysis of differentially expressed genes involved in immune response, carbon metabolism, apoptosis, and ribosomal function suggested that these may be key factors in the long-term adaptation to low-oxygen environments (Xing etal.2024).

5.1 Extractive Techniques

5.2 Separation and Purification Technology

5.3 Methods for Mining and Evaluation of Bioactive Ingredients

5.4 Food Development Strategy

Currently, no studies have confirmed whether the activity of P. esculenta affects the quality of bioactive substances in the body. Nonetheless, P. esculenta is anticipated to evolve into a functional food with medicinal properties. Maintaining ingredient freshness is crucial for optimal taste. Concerns also arise regarding the preservation of active ingredients during transportation, storage, and production of marketable products. Various storage technologies are available for this purpose. Ultra-high pressure technology, a non-thermal food preservation method, utilizes liquid as a pressure transfer medium to deactivate food-borne pathogens and enzymes in pressure-treated food. This technology has seen widespread adoption in the food industry in recent years (Wang etal.2016). For instance, treating frozen pink salmon fillets with ultra-high pressure technology minimally affects their odor while reducing the resistance of pathogens like Listeria monocytogenes and Salmonella enterica (Boziaris etal.2021). Méndez etal. observed that ultra-high pressure treatment inhibits lipid hydrolysis in sardines without altering the activity of enzymes such as acid phosphatase and cathepsin B and D, and without causing significant changes in sarcoplasmic and myofibrillar proteins (Méndez etal.2017). Given its pronounced seafood flavor, P. esculenta holds promise as a seafood seasoning. Even discarded parts like coelomic fluid can be repurposed. Utilizing the Maillard reaction to create condiments from P. esculenta and its by-products can recover nutrients from these by-products, enhance the value of P. esculenta, and introduce new products, thereby addressing issues of low product utilization to some extent (Wang etal.2019).