The Effects of Four Different Thawing Methods on Quality Indicators of Amphioctopus neglectus.

Amphioctopus neglectus is a species of octopus that is favored by consumers due to its rich nutrient profile. To investigate the influence of different thawing methods on the quality of octopus meat, we employed four distinct thawing methods: air thawing (AT), hydrostatic thawing (HT), flowing water thawing (FWT), and microwave thawing (MT). We then explored the differences in texture, color, water retention, pH, total volatile basic nitrogen (TVB-N), total sulfhydryl content, Ca2+-ATPase activity, and myofibrillar protein, among other quality indicators in response to these methods, and used a low-field nuclear magnetic resonance analyzer to assess the water migration that occurred during the thawing process. The results revealed that AT had the longest thawing time, leading to oxidation-induced protein denaturation, myofibrillar protein damage, and a significant decrease in water retention. Additionally, when this method was utilized, the content of TVB-N was significantly higher than in the other three groups. HT, to a certain extent, isolated the oxygen in the meat and thus alleviated protein oxidation, allowing higher levels of Ca2+-ATPase activity, sulfhydryl content, and springiness to be maintained. However, HT had a longer duration: 2.95 times that of FWT, resulting in a 9.84% higher cooking loss and a 28.21% higher TVB-N content compared to FWT. MT had the shortest thawing time, yielding the lowest content of TVB-N. However, uneven heating and in some cases overcooking occurred, severely damaging the protein structure, with a concurrent increase in thawing loss, W value, hardness, and shear force. Meanwhile, FWT improved the L*, W* and b* values of octopus meat, enhancing its color and water retention. The myofibrillar protein (MP) concentration was also the highest after FWT, with clearer subunit bands in SDS-PAGE electrophoresis, indicating that less degradation occurred and allowing greater springiness, increased Ca2+-ATPase activity, and a higher sulfhydryl content to be maintained. This suggests that FWT has an inhibitory effect on oxidation, alleviating protein oxidation degradation and preserving the quality of the meat. In conclusion, FWT outperformed the other three thawing methods, effectively minimizing adverse changes during thawing and successfully maintaining the quality of octopus meat.

Targeted Affinity Purification and Mechanism of Action of Angiotensin-Converting Enzyme (ACE) Inhibitory Peptides from Sea Cucumber Gonads.

Protein hydrolysates from sea cucumber (Apostichopus japonicus) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC50 value of 333.5 µmol·L-1, HDWWKER with an IC50 value of 583.6 µmol·L-1, and THDWWKER with an IC50 value of 1291.8 µmol·L-1) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔGtotal) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol-1, -71.6 Kcal·mol-1, and -69.1 Kcal·mol-1, respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.

Characteristics of hairtail surimi gels treated with myofibrillar protein-stabilized Pickering emulsions.

BACKGROUND: Hairtail (Trichiurus haumela) surimi exhibits poor gelation properties and a dark gray appearance, which hinder its utilization in high-quality surimi gel products. The effect of Pickering emulsions stabilized by myofibrillar proteins (MPE) on the gel properties of hairtail surimi has been unclear. In particular, the impact of MPE under NaCl and KCl treatments on the quality of hairtail surimi gels requires further elucidation. RESULTS: Pickering emulsions stabilized by myofibrillar proteins and treated with NaCl or KCl (Na-MPE, K-MPE) were added to hairtail surimi in amounts of 10-70 g kg-1. The addition of 50 g kg-1 Na-MPE and K-MPE improved the gel strength, textural properties, whiteness, and water-holding capacity (WHC) of hairtail surimi. The relative content of ß-turn and ß-sheet in the surimi gels increased and the relative content of random coils and α-helix decreased with the addition of oil. The addition of Na-MPE and K-MPE did not affect the secondary structure of surimi gels but stimulated the gelation of hairtail surimi gels. Hairtail surimi containing K-MPE demonstrated similar performance in terms of hardness, microstructure, and WHC compared with the addition of Na-MPE. CONCLUSION: The quality of hairtail surimi gels can be improved by the addition of Na-MPE or K-MPE. The K-MPE proved to be an effective option for enhancing the properties of hairtail surimi gels at 50 g kg-1 to replace Na-MPE. © 2024 Society of Chemical Industry.

Simultaneous ultrasound and microwave application in myosin-chlorogenic acid conjugation: Unlocking enhanced emulsion stability.

This study investigated the grafting chlorogenic acid (CA) onto myosin, utilizing various techniques including conventional method, ultrasound, microwave, and combination of ultrasound and microwave (UM). The grafting efficiency was as follows: conventional method < microwave < ultrasound < UM. The UM technique manifested the highest CA-binding capacity (80.26 µmol/g myosin) through covalent bonding, and a much shorter time was required for conjugation than conventional method. The conjugation of polyphenol significantly increased the solubility of myosin with reduced aggregation behavior, which was accompanied by structural alterations from ordered structures (α-helix and ß-sheet) to disordered forms. The emulsion stabilized by UM-myosin-CA conjugate exhibited the most homogeneous microstructure with favorable creaming stability. Moreover, the resulting emulsion presented strong oxidation resistance and storage stability. These results illustrate the promising potential of employing CA-grafted myosin, especially when processed using the UM technique, in the development of highly efficient emulsifiers.

Isolation and Purification of Protamine from the Cultured Takifugu flavidus and Its Physicochemical Properties.

Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable insulin and as a heparin antagonist, combatting fatigue, etc. Thus, it has been widely used in medicinal applications and food products. Cultured Takifugu flavidus is a type of pufferfish with a delicious taste that is popular in China, and its production is increasing significantly. Therefore, protamine was extracted via acid extraction from the sperm of Takifugu flavidus and further isolated and purified via sephadex gel chromatography, ion exchange chromatography, and desalination chromatography. Furthermore, the physicochemical properties of protamine were investigated. The results showed that the sperm of the cultured T. flavidus were non-toxic, and the extracted and purified protamine had high contents of arginine (36.90%) and lysine (27.02%), respectively. The secondary structure of protamine was mainly ß-folded and irregularly curled. Additionally, protamine exhibited high thermal stability with a denaturation temperature of 176 °C. This study would provide a theoretical basis for the structural analysis, bioactivity, and resource development of pufferfish protamine and help to promote the development of the pufferfish industry.

Advances in Research on Marine-Derived Lipid-Lowering Active Substances and Their Molecular Mechanisms.

Hyperlipidemia (HLP) is a metabolic disorder caused by abnormal lipid metabolism. Recently, the prevalence of HLP caused by poor dietary habits in the population has been increasing year by year. In addition, lipid-lowering drugs currently in clinical use have shown significant improvement in blood lipid levels, but are accompanied by certain side effects. However, bioactive marine substances have been shown to possess a variety of physiological activities such as hypoglycemic, antioxidant, antithrombotic and effects on blood pressure. Therefore, the hypolipidemic efficacy of marine bioactive substances with complex and diverse structures has also attracted attention. This paper focuses on the therapeutic role of marine-derived polysaccharides, unsaturated fatty acids, and bioactive peptides in HLP, and briefly discusses the main mechanisms by which these substances exert their hypolipidemic activity in vivo.

Exploring the Potential of Water-Soluble Squid Ink Melanin: Stability, Free Radical Scavenging, and Cd2+ Adsorption Abilities.

Squid ink melanin can be efficiently extracted from the byproduct ink sac generated during squid processing. As a natural food colorant, it possesses inherent antioxidant properties and the capability to adsorb heavy metals. This study aims to investigate the solubility of water-soluble squid ink melanin (WSSM) obtained from the ink sac, as well as its stability under various conditions including temperature, pH, salt, sugar, potassium sorbate, metal ions, sodium benzoate, sodium sulfite (reducing agent), and hydrogen peroxide (oxidizing agent). Moreover, it explores the scavenging effects of WSSM on free radicals and cadmium ions. The findings suggest that WSSM's stability is insignificantly affected by high temperature, sucrose, and salt. However, acidity, sodium benzoate, potassium sorbate, sodium sulfite (Na2SO3), and hydrogen peroxide (H2O2) significantly influence its stability. Most metal ions do not impact the stability of WSSM, except for Fe2+, Fe3+, Al3+, and Cu2+, which result in the precipitation of WSSM. Additionally, WSSM exhibits remarkable antioxidant activity with IC50 values of 0.91, 0.56, and 0.52 mg/mL for scavenging superoxide anion radicals (O2-·), hydroxyl radicals (·OH), and DPPH radicals, respectively. It also demonstrates the ability to adsorb the heavy metal Cd2+, with the adsorption rate gradually increasing with a higher temperature and larger amounts of WSSM added. Infrared spectroscopy analysis reveals the weakening of characteristic peaks (-COOH and -OH) during the process of Cd2+ adsorption by WSSM, while SEM confirms surface roughening and structural damage after Cd2+ adsorption. This study provides valuable insights for the utilization of squid melanin products as natural antioxidants and heavy metal adsorbents in the food industry.

Dynamic Changes in the Microbial Composition and Spoilage Characteristics of Refrigerated Large Yellow Croaker (Larimichthys crocea) during Storage.

The quality changes, dynamic changes in microbial composition, and diversity changes in large yellow croaker (Larimichthys crocea) during 4 °C refrigeration were studied using 16S rDNA high-throughput sequencing technology, and the total viable count (TVC), total volatile basic nitrogen (TVB-N), and thiobarbituric acid-reactive substances (TBARS) were determined. The results revealed a consistent increase in TVC, TVB-N, and TBARS levels over time. On the 9th day, TVC reached 7.43 lg/(CFU/g), while on the 15th day, TVB-N exceeded the upper limit for acceptable quality, reaching 42.56 mg/100 g. Based on the 16S rDNA sequencing results, we categorized the storage period into three phases: early storage (0th and 3rd days), middle storage (6th day), and late storage (9th, 12th, and 15th days). As the storage time increased, both the species richness and diversity exhibited a declining trend. The dominant genus identified among the spoilage bacteria in refrigerated large yellow croaker was Pseudomonas, accounting for a high relative abundance of 82.33%. A comparison was carried out of the spoilage-causing ability of three strains of Pseudomonas screened and isolated from the fish at the end of storage, and they were ranked as follows, from strongest to weakest: P. fluorescen, P. lundensis, and P. psychrophila. This study will provide a theoretical basis for extending the shelf life of large yellow croaker.

In vitro-simulated intestinal flora fermentation of Porphyra haitanensis polysaccharides obtained by different assisted extractions and their fermented products against HT-29 human colon cancer cells.

Herein, we studied the in vitro-simulated intestinal flora fermentation of Porphyra haitanensis polysaccharides (PHPs) with microwave, ultrasonic, ultra-high pressure-assisted extraction and the protective effect of their fermented products against HT-29 human colon cancer cells. The results showed that PHPs were largely degraded at the 18 h stage of ascending colon fermentation, further greatly increasing the contents of reducing sugars and short-chain fatty acids (p < 0.05). Particularly, the PHPs subjected to ultra-high pressure-assisted extraction (UHP-PHP) showed the highest reducing sugar content of 1.68 ± 0.01 mg mL-1 and butyric acid content of 410.77 ± 7.99 mmol mL-1. Moreover, UHP-PHP showed a better effect in increasing the ratio of Bacteroidetes/Firmicutes and decreasing the abundance of Proteobacteria and Escherichia coli. PHPs could protect against HT-29 cells by increasing the ROS levels in a concentration-dependent manner, especially UHP-PHP fermented in a descending colon for 24 h. This was related to the up-regulated apoptosis-related genes (Bax and Bak), down-regulated protein expression of Bcl-2 and activation of the p-AKT protein, thereby promoting the apoptosis of HT-29 cells. Our results can facilitate the modification of PHPs and their practical application in the development of intestinal health improving products.

Affinity Purification and Molecular Characterization of Angiotensin-Converting Enzyme (ACE)-Inhibitory Peptides from Takifugu flavidus.

An affinity chromatography filler of CNBr-activated Sepharose 4B-immobilized ACE was used to purify ACE-inhibitory peptides from Takifugu flavidus protein hydrolysate (<1 kDa). Twenty-four peptides with an average local confidence score (ALC) ≥ 80% from bounded components (eluted by 1 M NaCl) were identified by LC-MS/MS. Among them, a novel peptide, TLRFALHGME, with ACE-inhibitory activity (IC50 = 93.5 µmol·L-1) was selected. Molecular docking revealed that TLRFALHGME may interact with the active site of ACE through H-bond, hydrophobic, and electrostatic interactions. The total binding energy (ΔGbinding) of TLRFALHGME was estimated to be -82.7382 kJ·mol-1 by MD simulations, indicating the favorable binding of peptides with ACE. Furthermore, the binding affinity of TLRFALHGME to ACE was determined by surface plasmon resonance (SPR) with a Kd of 80.9 µmol, indicating that there was a direct molecular interaction between them. TLRFALHGME has great potential for the treatment of hypertension.

Structural properties and emulsification of myofibrillar proteins from hairtail (Trichiurus haumela) at different salt ions.

The structural properties and emulsification of myofibrillar proteins (MPs) are susceptibly affected by salt ions. The effect of different salt ions on the structural properties and emulsification of MPs from hairtail (Trichiurus haumela) remains unclear. Hairtail MPs were analyzed under different ion treatments of Na+, K+, Ca2+ and Mg2+. MPs at K+ and Na+ treatment showed a similar trend on salt effect due to the unfolding of proteins under salt ions. However, the excessive electrostatic effect of divalent ions could enhance protein aggregation, especially at Ca2+ and Mg2+. The ß-sheet of MPs at different salt ions interconverted with α-helix and random coil at ionic strengths from 0.1 mol/L to 1.0 mol/L. The surface hydrophobicity and active sulfhydryl content of MPs increased with the improvement of ionic strengths at 0-0.8 mol/L. Under Ca2+ and Mg2+ treatments, the turbidity of MPs was low compared to that under the treatment of Na+ and K+. Additionally, the emulsification of hairtail MPs treated with different ions was improved at an ionic strength of 0.6 mol/L. This study can contribute to using salts in constructing fish protein-based emulsions for manufacturing emulsified surimi products and promoting the development and utilization of hairtail proteins.

Structural characterization and antioxidant activity of polysaccharides extracted from Porphyra haitanensis by different methods.

This study was to investigate the structure and antioxidant activity of Porphyra haitanensis polysaccharides (PHPs) extracted by different methods, including water extraction (PHP), ultra-high pressure (UHP-PHP), ultrasonic (US-PHP) and microwave assisted water extraction (M-PHP). Compared with water extraction, the total sugar, sulfate and uronic acid contents of PHPs was enhanced by ultra-high pressure, ultrasonic and microwave assisted treatments, especially those of UHP-PHP were increased by 24.35 %, 12.84 % and 27.51 %, respectively (p < 0.05). Meanwhile, these assisted treatments affected the monosaccharide ratio of polysaccharides and significantly reduced the protein content, molecular weight as well as particle size of PHPs (p < 0.05), and resulted in a loose microstructure with more porosity and fragments. PHP, UHP-PHP, US-PHP, and M-PHP all possessed in vitro antioxidant capacity. Among them, UHP-PHP had the strongest oxygen radical absorbance capacity, DPPH and ·OH radicals scavenging capacities, which increased by 48.46 %, 116.24 %, and 14.98 % respectively. Moreover, PHPs particularly UHP-PHP effectively increased the cell viability and reduced ROS levels of H2O2 induced RAW264.7 cells (p < 0.05), indicating their good effects against cell oxidative damage. The findings suggested that PHPs with ultra-high pressure assisted treatments has the better potential to develop natural antioxidant.

Characteristics and potential application of myofibrillar protein from golden threadfin bream (Nemipterus virgatus) complexed with chitosan.

The strategies to broaden the applications of proteins involve their modification with polysaccharides. However, the characteristics and application of myofibrillar proteins (MPs) from golden threadfin bream (Nemipterus virgatus) complexed with chitosan (CS) are still unclear. Therefore, the characteristics of MPs complexed with CS and their application were investigated at different MP/CS ratios (100:0-80:20 (w/w)). The turbidity of MP/CS complexes was small at the MP/CS ratio of 95:5 (w/w). Besides, CS addition induced changes in MP structure to make it hydrophilic. Secondary structure analysis showed that α-helix and ß-turn interconverted with ß-sheet and random coil after the addition of CS. Additionally, the thermal stability of MP/CS mixtures enhanced after the addition of CS and the MP/CS mixtures at the ratio of 95:5 (w/w) had a relatively compact structure. High internal phase emulsions (HIPEs) prepared at the MP/CS ratio of 95:5 (w/w) were relatively stable compared to those at the other ratios. Consequently, MP/CS mixtures at suitable ratios possess the potential ability to prepare HIPEs. These results exhibit that MP/CS mixtures may be applied for constructing food-graded emulsion delivery systems with a high internal phase in the food industry.

Application of polysaccharide-rich solution derived from waste macroalgae Enteromorpha prolifera in cherry tomato preservation and utilizing post-extraction residue for crude bio-oil production.

Preservative is of importance to retard fruit deterioration and prolong the shelf-life. The suitability of using water-soluble polysaccharide extracted from waste macroalgae Enteromorpha prolifera (EPP) for cherry tomato preservation was evaluated. As compared with the control, the EPP-treated cherry tomatoes exhibited better fruit appearance, lower disease index and rot index during storage. Around 47 % EPP-treated cherry tomatoes were commercially acceptable after 36 days, which was however only 15.6 % for untreated cherry tomatoes, indicating the satisfactory preservation effectiveness of EPP-rich solution for cherry tomatoes. The post-extraction residue was commonly underutilized, we herein attempted to employ an emerging thermochemical conversion technique, hydrothermal liquefaction, to produce crude bio-oil (biocrude) from post-extraction E. prolifera. A biocrude yield of ∼23 wt% (dry-ash-free, daf) was obtained, and fatty acids and phenolics were identified to be the two main components in biocrude. The biocrude contained ∼70 % carbon and the higher heating value was ∼30 MJ/kg.

Screening and Molecular Mechanisms of Novel ACE-Inhibitory Peptides from Gracilariopsis lemaneiformis.

Candidate peptides with novel angiotensin-I-converting enzyme (ACE) inhibitor activity were obtained from hydrolysates of Gracilariopsis lemaneiformis by virtual screening method. Our results showed that G. lemaneiformis peptides (GLP) could significantly lower blood pressure in spontaneously hypertensive rats (SHR). At least 101 peptide sequences of GLP were identified by LC-MS/MS analysis and subjected to virtual screening. A total of 20 peptides with the highest docking score were selected and chemically synthesized in order to verify their ACE-inhibitory activities. Among them, SFYYGK, RLVPVPY, and YIGNNPAKG showed good effects with IC50 values of 6.45 ± 0.22, 9.18 ± 0.42, and 11.23 ± 0.23 µmoL/L, respectively. Molecular docking studies revealed that three peptides interacted with the active center of ACE by hydrogen bonding, hydrophobic interactions, and electrostatic forces. These peptides could form stable complexes with ACE. Furthermore, SFYYGK, RLVPVPY, and YIGNNPAKG significantly reduced systolic blood pressure (SBP) in SHR. YIGNNPAKG exhibited the highest antihypertensive effect, with the largest decrease in SBP (approximately 23 mmHg). In conclusion, SFYYGK, RLVPVPY, and YIGNNPAKG can function as potent therapeutic candidates for hypertension treatment.

Molecular Characterization of a New Tetrodotoxin-Binding Protein, Peroxiredoxin-1, from Takifugu bimaculatus.

Pufferfish are considered a culinary delicacy but require careful preparation to avoid ingestion of the highly toxic tetrodotoxin (TTX), which accumulates in certain tissues. In this study, the tissue distribution of peroxiredoxin-1 from Takifugu bimaculatus was investigated. The peroxiredoxin-1 protein was obtained by in vitro recombinant expression and purification. The recombinant protein had a strong ability to scavenge hydroxyl radicals, protect superhelical DNA plasmids from oxidative damage, and protect L929 cells from H2O2 toxicity through in vitro antioxidant activity. In addition, we verified its ability to bind to tetrodotoxin using surface plasmon resonance techniques. Further, recombinant proteins were found to facilitate the entry of tetrodotoxin into cells. Through these analyses, we identified, for the first time, peroxiredoxin-1 protein from Takifugu bimaculatus as a potential novel tetrodotoxin-binding protein. Our findings provide a basis for further exploration of the application of peroxiredoxin-1 protein and the molecular mechanisms of tetrodotoxin enrichment in pufferfish.

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus.

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1-3, 3-10, 10-50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC-MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC50 of 28 µmmol·L-1). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.

Novel polysaccharide extracted from Sipunculus nudus inhibits HepG2 tumour growth in vivo by enhancing immune function and inducing tumour cell apoptosis.

A novel polysaccharide was extracted from Sipunculus nudus (SNP). The molecular weight (MW) of SNP was determined to be 9223 Da by high-performance gel permeation chromatography analyses, and the structure of the SNP repeat units was determined to be →3,4-ß-D-GlcpNAC (1→ and →4) -α-D-Glcp (1→ in the ratio of 15:1; →2) -α -D-Galp - (1→ as a side chain; and ß-D-Galp-(1→ and α- D-Glcp - (1→ as end groups by GC-MS analysis and NMR assays. The effect of SNP on hepatoma HepG2-bearing mice was analysed to verify its potential in the clinical treatment of liver cancer. A total of 90 male athymic nu/nu mice were divided into therapeutic and preventive groups and fed with different amounts of SNP. The antitumour effect of SNP on HepG2-bearing mice and mechanism of such were studied by analysing the tumour size, spleen index, thymus index, immune factors in the blood, tumour apoptosis factors, etc. The results suggest that SNP not only increased the index of immune organs in the body, but also enhanced the secretion of immune factors, including interleukin-2, interferon gamma and tumour necrosis factor-alpha in the serum. SNP induced the apoptosis of tumour cells via the mitochondrial apoptosis pathway, which upregulated caspase-3, caspase-8, caspase-9 and BCL2-associated X, but downregulated B-cell lymphoma-2 and vascular endothelial growth factor protein expression. In conclusion, SNP inhibited tumour growth by enhancing immune function and inducing tumour cell apoptosis in HepG2-bearing mice. Therefore, SNP may be further investigated as a promising candidate for future antitumour drugs.

Molecular characterization, purification, and antioxidant activity of recombinant superoxide dismutase from the Pacific abalone Haliotis discus hannai Ino.

Superoxide dismutase (SOD) is an acidic metalloenzyme that scavenges free radicals produced by endogenous and exogenous substances. In the present study, the tissue distribution of the superoxide dismutase HdhCu/Zn-SOD was investigated in Haliotis discus hannai Ino. The expression profile after lipopolysaccharide (LPS) challenge was determined using quantitative real-time polymerase chain reaction (qPCR). To study the antioxidant activity of a recombinant HdhCu/Zn-SOD protein, the HdhCu/Zn-SOD gene was cloned into the pPIC9K vector and transformed into the Pichia pastoris GS115 strain by electroporation. After induction by methanol, the recombinant product was purified using immobilized metal affinity chromatography and confirmed using mass spectrometry. The optimal expression conditions were determined to be incubation with 0.5% methanol at pH 6.0, resulting in a stable expressed product with the molecular weight of approximately 17 kDa and 21 kDa. The enzymatic activity of HdhCu/Zn-SOD consistently increased with increasing Cu2+ concentrations and showed good thermal stability. Recombinant HdhCu/Zn-SOD showed a strong ability to scavenge superoxide anions and hydroxyl radicals and protected L929 cells against the toxicity caused by H2O2 through its in vitro antioxidant activity. The heterologous expression of HdhCu/Zn-SOD in P. pastoris and the antioxidant activity of this enzyme are reported for the first time.