Improving the in vitro and in vivo bioavailability of pterostilbene using Yesso scallop gonad protein isolates-epigallocatechin gallate (EGCG) conjugate-based emulsions: effects of carrier oil.

This study investigated the influence of carrier oils on the in vitro and in vivo bioavailability of PTE encapsulated in scallop gonad protein isolates (SGPIs)-epigallocatechin gallate (EGCG) conjugate stabilized emulsions. The SGPIs-EGCG stabilized emulsions were subjected to an in vitro simulated digestion, and the resulting corn oil and MCT micelles were used to evaluate the PTE transportation using the Caco-2 cell model. Both emulsions remarkably improved the bioaccessibility of PTE in the micelle phase. Nevertheless, corn oil emulsions increased trans-enterocyte transportation of PTE more efficiently than MCT emulsions. Furthermore, the maximum plasma concentrations of PTE and its metabolites in mice fed with PTE emulsions were prominently higher than those in mice fed with PTE solution, while the in vivo metabolic patterns of PTE in different oil-stabilized emulsions were different. Therefore, SGPIs-EGCG stabilized emulsions could enhance the bioavailability of PTE through controlled release, in which corn oil is more suitable than MCT.

Formation and stability of electrostatic complexes formed between scallop female gonad protein isolates and sodium alginate: Influence of pH, total concentration, blend ratio, and ionic strength.

The complex coacervation between scallop (Patinopecten yessoensis) female gonad protein isolates (SFGPIs) and sodium alginate (SA) was determined by the turbidimetric method. The impact of pH, total biopolymer concentration, biopolymer blend ratio, and various salt ionic on the mechanisms governing the complex coacervation of SFGPIs-SA complexes were also investigated. For the SFGPIs:SA ratio of 2:1 without adding NaCl, insoluble and soluble complexes were observed at pH 5.8 (pHφ1 ) and pH 8.2 (pHc ) with the optimum biopolymer interactions appearing at pH 2.6 (pHopt ). The maximum turbidity value increased with the increment of the total biopolymer concentration from 0.37 to 1.83 until attaining the critical value (0.75%). As the blend ratios rose from 1:3 to 12:1, the critical pH values (pHc , pHφ1 , and pHopt ) moved to higher pH. Furthermore, the addition of NaCl led to a remarkable decrease in turbidity over the whole pH region in SFGPIs-SA complexes. Moreover, monovalent ions (Na+ and K+ ) had the same effect on the formation of the SFGPIs-SA complex, whereas the divalent cations (Mg2+ and Ca2+ ) lessened the complex formation in comparison with the monovalent ions. This study offers a methodological and theoretical basis for the design of complex SFGPIs-SA systems by understanding the complex coacervation under different conditions. PRACTICAL APPLICATION: In recent years, several protein-polysaccharides complexes have been widely applied in food and biological systems. Scallop (Patinopecten yessoensis) female gonads are deemed as good marine sources for developing protein matrices on account of their high protein content and nutrients. In our study, the effects of different conditions on the mechanisms governing the complex coacervation of SFGPI-SA mixtures were investigated, and the instability of the system could be overcome by understanding the conditions for SFGPIs/SA complex formation, which have a feasible role in developing marine source-protein as a functional food base such as kamaboko gels, can, sausage, fat substitutes, and delivery vehicles for bioactive compounds.

Quantitative proteomics reveals the relationship between protein changes and off-flavor in Russian sturgeon (Acipenser gueldenstaedti) fillets treated with low temperature vacuum heating.

This study aimed to reveal the molecular mechanisms associated with off-flavor generation in sturgeon fillets treated by low temperature vacuum heating (LTVH). Label-free quantitative proteomics was used to identify 120 favor-related proteins, 27 proteins were screened as differentially expressed for bioinformatics analysis. 17 of KEGG pathways were identified. Particularly, proteins involved in proteasome and peroxisome were highly correlated with off-flavor formation. They were primarily implicated in the structures of proteins, including binding and proteasome pathways. The results indicated that the LTVH reduced the binding sites by down-regulating protease and superoxide dismutase expression. LTVH increased the myofibrillar protein and sulfhydryl content and decreased the total volatile basic nitrogen and thiobarbituric acid reactive substance, which confirmed that protein oxidation was related to off-flavor. This proteomics study provided new insights into the off-flavor of sturgeon with LTVH, and proposed potential link between biological processes and off-flavor formation.

Structural characteristics and improved in vitro hepatoprotective activities of Maillard reaction products of decapeptide IVTNWDDMEK and ribose.

Here, a novel decapeptide IVTNWDDMEK with Maillard reactivity derived from scallop Chlamys farreri mantle was identified. The structural characteristics and in vitro hepatoprotective effects of IVTNWDDMEK conjugated with ribose were further investigated. The changes in decapeptide structures were determined by ultraviolet-visible (UV-vis), Fourier transform infrared (FTIR), and atomic force microscopy (AFM), and the modification sites induced by Maillard reaction of IVTNWDDMEK and ribose were monitored by high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Maillard reaction products (MRPs) of IVTNWDDMEK-ribose demonstrate hepatoprotective benefits through the suppression of DNA damage and apoptosis induced by oxidative stress in human HepG2 cells in addition to enhancing the antioxidant activities. Moreover, after treatment with decapeptide-ribose MRPs, the activities of cellular antioxidative enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Rx) were remarkably increased, while the content of malondialdehyde (MDA) was decreased compared with H2 O2 - treated group, thereby enhancing the intracellular antioxidant defenses. These findings demonstrate the potential utilization of decapeptide IVTNWDDMEK-ribose MRPs as food antioxidants to suppress oxidative damage. PRACTICAL APPLICATION: In recent years, several food-derived bioactive peptides and their derivatives are regarded as good dietary antioxidants for reducing oxidative stress and improving liver function. Here, a novel Maillard reactive decapeptide IVTNWDDMEK, identified from scallop mantle hydrolysates by peptidomics in the previous study was synthesized. Then, the correlation between intercellular antioxidant activities and chemical structure changes of IVTNWDDMEK-ribose Maillard reaction conjugates was further studied. The preferable hepatoprotective activities of decapeptide IVTNWDDMEK-ribose MRPs indicated that these MRPs could be potentially utilized as food antioxidants or additives in the production of nutritional foods.

Conjugation of (-)-epigallocatechin-3-gallate and protein isolate from large yellow croaker (Pseudosciaena crocea) roe: improvement of antioxidant activity and structural characteristics.

BACKGROUND: Large yellow croaker (Pseudosciaena crocea) roe is the main by-product in the processing of large yellow croaker. Previous studies have found that its protein isolates are composed of vitellogenin, as well as vitellogenin B and C, having good functional properties. (-)-Epigallocatechin-3-gallate (EGCG) is a natural antioxidant component that can be combined with protein to improve antioxidant activity and structural characteristics of protein. RESULTS: EGCG was bound with the P. crocea roe protein isolate (pcRPI) by the free radical method to prepare the conjugate. The formation of pcRPI-EGCG conjugates was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography, which showed that the calculated weight-average molar masses of native-pcRPI and pcRPI-EGCG conjugates were 86.9 and 215.3 kDa, respectively. The results of fluorescence, ultraviolet, circular and infrared spectra indicated that the conjugation of EGCG with native-pcRPI changed the secondary and tertiary structure of native-pcRPI. The pcRPI-EGCG conjugates exhibited higher thermal stability than native-pcRPI. The radical scavenging and reducing power of native-pcRPI were increased by 2.0-2.5- and 1.4-fold, respectively, after the EGCG-grafting reaction. CONCLUSION: These results indicate that the binding of pcRPI and EGCG effectively improved the antioxidant properties and structural characteristics of the pcRPI. © 2021 Society of Chemical Industry.

Intermolecular interaction in the hybrid gel of scallop (Patinopecten yessoensis) male gonad hydrolysates and κ-carrageenan.

Various bond disrupting agents including NaCl, GuHCl, urea, and SDS were introduced to investigate the intermolecular interactions between scallop (Patinopecten yessoensis) male gonad hydrolysates (SMGHs) and κ-carrageenan (κ-C), which were monitored by changes in rheological property, water distribution, conformation characterization and microstructure by using rheometer, low field-NMR relaxometry, Fourier transform infrared (FTIR) spectroscopy, cryo-scanning electron microscopy (cryo-SEM), and confocal laser scanning microscopy. The results showed that the bond disrupting agents deteriorated the rheological property of SMGHs/κ-C in a dose-dependent manner. Indeed, at the same concentration of 2 M, NaCl deteriorated the SMGHs/κ-C more obviously than GuHCl and urea. In addition, SMGHs/κ-C with bond disrupting agents possessed higher relaxation times including T21 and T23 , indicating the migration to free water direction of bound and free water. Moreover, the FITR results showed the red-shift in water regions (amide A and B bands), amide I and II bands, and indicated the breakdown of hydrogen bonds and electrostatic interactions, indicating a disordered structure in SMGHs/κ-C by various bond disrupting agents. Furthermore, cryo-SEM results showed the change of SMGHs/κ-C from a homogeneous network to a looser and ruptured one with larger void spaces, and indicated the disrupted and tattered microstructure of SMGHs/κ-C by various bond disrupting agents. Additionally, SMGHs/κ-C as well showed less aggregates stained by RITC by bond disrupting agents. These results suggest that electrostatic interactions would be mainly involved in the maintenance of SMGHs/κ-C gel network. This study could provide theoretical and methodological basis for hydrogel products with modified gel strength and microstructure by understanding the intermolecular interactions in gel system. PRACTICAL APPLICATION: Scallop (Patinopecten yessoensis) male gonads as a high-protein part of scallop, is usually discarded during processing despite its edibility. In recent years, scallop male gonads are regarded as good sources to develop protein matrices due to their high protein content and numerous nutrients. In this study, scallop male gonad hydrolysates (SMGHs) were obtained by trypsin-treated process. The considerable gelation behavior of SMGHs indicated that the SMGHs could be potentially utilized as a novel thickener and additive in production of kamaboko gels, can, sausage and spread with marine flavor.

Effect of pH and mixing ratio on interpolymer complexation of scallop (Patinopecten yessoensis) male gonad hydrolysates and κ-carrageenan.

The electrostatic complex coacervation between scallop Patinopecten. yessoensis male gonad hydrolysates (SMGHs) and κ-carrageenan (κ-C) were monitored by using turbidimetry at various pH (1-12) and biopolymer mixing ratio (9:1-1:9). The pHc exhibited ratio-independent behavior, and pHφ1, pHmax exhibited ratio-dependent behavior, respectively. The decreasing ratio enhanced the gel strength of SMGHs/κ-C at higher pH while inversely at lower pH, ascribing to more SMGHs aggregates and stronger neutralization between positively charged patches in SMGHs and κ-C at lower pH and higher ratio. Moreover, SMGHs/κ-C gel at acid condition exhibited lower relaxation times (T21 and T23). Furthermore, the rheological and relaxation time T2 data were well associated with microscopy images which indicated that SMGHs/κ-C gel showed a well-distributed network structure at more acidic domains, supporting stronger gel rigidity and water-holding capacity.

Complex coacervation of scallop (Patinopecten yessoensis) male gonad hydrolysates and κ-carrageenan: Effect of NaCl and KCl.

The complex coacervation between scallop Patinopecten yessoensis male gonad hydrolysates (SMGHs) and κ-carrageenan (κ-C) as influenced by ionic strength (NaCl and KCl) were monitored by using turbidimetric analysis at pH 1-12. The optical density of SMGHs/κ-C complexes initially increased at lower ionic strength (0-0.5 M) whereas decreased at higher ionic strength (0.5-1.5 M) as a result of the salt-enhanced and salt-reduced effect, respectively. Both of pHc and pHφ1 exhibited ionic strength-dependent behavior that firstly shifted to acidic pH followed by an increase as salt increasing. Moreover, salt addition strongly promoted the gelation of SMGHs/κ-C which was mainly driven by electrostatic forces, as reflected by increasing storage modulus G' from 3621 to 6559 Pa, 2681-25631 Pa at 0.1 Hz and decreasing T23 relaxation time from 349.10 to 296.89 ms, 241.07-186.89 ms by 0-0.5 M NaCl/KCl, respectively. Furthermore, the rheological and relaxation time T2 data were well associated with microscopy images that SMGHs/κ-C gels with NaCl/KCl showed a denser network with more flocculation formation and larger pore sizes with thicker network wall, especially in KCl group, which indeed supported the gel elasticity. Taken together, this study could provide theoretical and methodological basis for new functional hydrogel products with modified gel strength and microstructure by understanding the complex coacervation in gel system.

Functional properties of gonad protein isolates from three species of sea urchin: a comparative study.

Sea urchin Mesocentrotus nudus, Glyptocidaris crenularis, and Strongylocentrotus intermedius gonad protein isolates (mnGPIs, gcGPIs, and siGPIs) were extracted by isoelectric solubilization/precipitation (ISP) from the defatted gonads, and their functional properties were compared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed the similar protein pattern between each protein isolate and defatted gonad, indicating the high efficiency of ISP processing for protein recovery. Amino acid profileconfirmed that the mnGPIs and siGPIs could be potential sources of essential amino acid in nature. As regard to functional properties, mnGPIs showed higher water- and oil- holding capacities followed bysiGPIs and gcGPIs and all protein isolates presented great foaming property. As for emulsifying activity index (EAI), mnGPIs, gcGPIs, and siGPIs showed the minimum solubility and EAI at pH 5, 3, and 4, respectively, and behaved a pH-dependent manner. The gcGPIs revealed the highest EAI from pH 6 to 8 among the samples. In addition, circular dichroism showed increased content of ß-sheet at the expense of α-helix and ß-turn, suggesting the structure denaturation of the protein isolates. Indeed, no statistical difference was observed between secondary structure of mnGPIs and siGPIs. Moreover, ISP processing increased free sulfhydryl content of sea urchin protein isolates, but no difference was observed among the samples. Furthermore, siGPIs revealed the highest amount of total sulfhydryl and disulfide bonds, whereas both defatted gonads and protein isolates from G. crenularis presented the maximum surface hydrophobicity. These results suggest that gonad protein isolates from three species of sea urchin possess various functionalities and therefore can be potentially applied in food system. PRACTICAL APPLICATION: Sea urchin M. nudus, G. crenularis, and S. intermedius gonads are edible, whereas the functional properties of protein isolates from sea urchin gonad remain unknown. In this case, the extraction and comparison of three species of sea urchin gonad protein isolates will not only confirm functional properties but also screen food ingredients with suitable functions. In this study, functionalities of protein isolates derived from M. nudus, G. crenularis, and S. intermedius gonads would provide potential application in bakery food and meat products or as emulsifier candidates in food system.

Simultaneous extraction by acidic and saline solutions and characteristics of the lipids and proteins from large yellow croaker (Pseudosciaena crocea) roes.

The objective of this study was to simultaneously obtain protein isolates and lipids from the dried powder of large yellow croaker (Pseudosciaena crocea) roes (pcRs) to achieve high-value utilization. Protein isolates and lipids were extracted simultaneously from pcRs by saline and acidic solutions. The purity of the protein isolates from the pcRs (pcRPIs) was greater than 70%, with vitellogenin, vitellogenin B and vitellogenin C as the main proteins. The lipids from pcRs (pcRLs) were mainly composed of triglycerides with high levels of EPA and DHA. The pcRPIs exhibited a higher surface hydrophobicity, water/oil holding capacity and emulsifying ability than those of the pcRs. Moreover, pcRPIs had a better oil holding capacity and emulsifying ability than soy protein isolate. These results suggest that protein isolates and lipids can be simultaneously extracted by saline and acidic solutions, and pcRPIs and pcRLs can be used as functional materials in the food industry.

Quantitative Proteome Reveals Variation in the Condition Factor of Sea Urchin Strongylocentrotus nudus during the Fishing Season Using an iTRAQ-based Approach.

To investigate the variation in the condition factor of the sea urchin Strongylocentrotus nudus (S. nudus), gonads were collected in May (MAY), June (JUN), and July (JUL), at the beginning (AUG-b) and end of August (AUG-e). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection of the gonads revealed an obvious enhancement of the band at about 37 kDa from July, which was identified as transforming growth factor-beta-induced protein ig-h3 (TGFBI) by nanoLC-ESI-MS/MS. Gonadal proteins were identified by isobaric tagging for relative and absolute quantitation (iTRAQ), and regulation of the identified proteins in pairs of the collected groups was observed. A total of 174 differentially expressed proteins (DEPs) were identified. Seven of the DEPs showed significant correlations with both the gonad index (GI) and protein content. These correlations included 6-phosphogluconate dehydrogenase, decarboxylating isoform X2 (6PGD), CAD protein, myoferlin isoform X8, ribosomal protein L36 (RL36), isocitrate dehydrogenase [NADP], mitochondrial isoform X2 (IDH), multifunctional protein ADE2 isoform X3, sperm-activating peptides (SAPs) and aldehyde dehydrogenase, and mitochondrial (ALDH). However, TGFBI had no correlation with gonad index (GI) or protein content. 6PGD, IDH, multifunctional protein ADE2 isoform X3, and ALDH were shown to interact with each other and might play key roles in changing the condition factor of S. nudus gonads.

Involvement of DNA in Gel Formation of Scallop (Patinopecten yessoensis) Male Gonad Hydrolysates and Corresponding Hybrid Gel with κ-Carrageenan.

Involvement of DNA in gelation and microstructural properties of scallop (Patinopecten yessoensis) male gonad hydrolysates (SMGHs) and corresponding hybrid gel with κ-carrageenan (SMGHs/κ-C) was studied using DNase pretreatment. Although DNase pretreatment significantly transformed SMGHs from weak gels to liquid, it made SMGHs have a superior synergistic effect on gel formation with κ-C by evidence of 2.7-fold G' and 1.1-fold melting temperature. However, the relaxation time (T21 and T23), functional groups, and flocculation behavior were comparable between SMGHs/κ-C and SMGHs/DNase/κ-C. Moreover, SMGHs/DNase/κ-C exhibited a denser network with more numerous patches and larger void spaces. These results suggest that DNA contributes to the gel formation of SMGHs whereas restricts more cationic peptides in SMGHs to bind sulfate groups in κ-C during gel formation.

Physiochemical Properties and Functional Characteristics of Protein Isolates from the Scallop (Patinopecten yessoensis) Gonad.

Protein isolates were recovered from scallop (Patinopecten yessoensis) gonads to develop a novel functional matrix by investigating their physiochemical and functional properties. Scallop gonad protein isolates (SGPIs) were prepared from degreased scallop gonads (DSGs) by an alkali extraction and isoelectric solubilization/precipitation (ISP) process. The protein compositions of the SGPIs were mainly vitellogenin and beta-actin with molecular weights of 266 and 42 kDa, respectively, as determined using Nano-liquid chromatography-mass/mass (Nano-LC-MS/MS). After the ISP process, the protein solubility of the SGPIs was significantly improved, and the surface hydrophobicity of SGPIs intensely increased by 1.1-fold, which were attributed to the exposure of aromatic residues such as phenylalanine, tyrosine, and tryptophan. However, the content of total/reactive sulfhydryl in SGPIs was decreased compared with that of DSGs. Meanwhile, the ISP process caused partial protein unfolding, as indicated by circular dichroism analysis, which exhibited a remarkable rise in the ß-sheet content with a parallel decline in the α-helix and random coil contents (P < 0.05). SGPIs exhibited a better oil absorption capacity and foaming property than both DSGs and soybean protein isolates (SPIs). Moreover, the emulsifying capacity of SGPIs was greatly enhanced by the ISP process, which was superior to the effect of commercial SPIs and was ascribed to its favorable solubility as well as surface characteristics. PRACTICAL APPLICATION: During the processing of scallop (Patinopecten yessoensis) adductors, scallop gonad, a high-protein part, is usually discarded as processing by-products despite its edibility. In recent years, scallop gonads are regarded as good sources to develop protein matrices due to their high protein content and numerous nutrients. In this study, scallop gonad protein isolates (SGPIs) were isolated by isoelectric solubilization/precipitation (ISP) process. The preferable solubility, foaming property coupled with high emulsifying property of SGPIs indicated that the SGPIs could be potentially utilized as a good protein emulsifier and additives in production of kamaboko gels, hamburger patties, sausages, and pet foods.

Bioaccessibility and cellular uptake of ß-carotene in emulsion-based delivery systems using scallop (Patinopecten yessoensis) gonad protein isolates: effects of carrier oil.

Emulsion-based delivery systems were structured by using scallop gonad protein isolates (SGPIs) as novel food-grade emulsifiers. The effects of carrier oil, including the long chain triglycerides (LCT) and medium chain triglycerides (MCT), on the bioaccessibility and cellular uptake of ß-carotene (BC) were investigated. Both LCT and MCT delivery systems remained stable at pH 7-8 but aggregated at lower pH values (3-6) according to the results of light scattering and microscopy measurements. LCT droplets fabricated within SGPIs were digested and released more slowly than MCT droplets during the simulated gastrointestinal tract digestion. The LCT emulsion showed higher BC bioaccessibility (65.5%) than the MCT emulsion (23.1%) as a result of the greater solubilization of BC in mixed micelles fabricated from long-chain fatty acids. Moreover, the LCT emulsion produced higher cellular uptake of BC as compared with the MCT emulsion in intestinal epithelial cells. These results demonstrated that SGPIs could be used as novel food-grade emulsifiers to protect lipophilic bioactive compounds in emulsion-based delivery systems, in which LCT is more suitable to encapsulate and deliver BC than MCT.

In silico assessment and structural characterization of antioxidant peptides from major yolk protein of sea urchin Strongylocentrotus nudus.

Sea urchin gonads have been demonstrated to contain major yolk protein (MYP), which can be hydrolyzed by enzymes to release biologically active peptides. The in silico analysis of the MYP sequence in the BIOPEP database showed the presence of fragments with antioxidant activity. The sequence was hydrolyzed by 21 kinds of proteases and 23 antioxidant peptides were obtained. Eight peptides, including Leu-Trp (LW), Arg-Trp (RW), Ala-Trp (AW), Thr-Trp (TW), Ala-Asp-Phe (ADF), Leu-Trp-Lys (LWK), Ser-Asp-Phe (SDF) and Leu-Tyr (LY), were screened and a score over 0.5 was obtained using PeptideRanker. The peptides LW, TW and LWK showed a stronger antioxidant capacity with IC50 values of 8.85, 9.59 and 9.62 mmol L-1, respectively, compared to that of glutathione (10.81 mmol L-1). Furthermore, AW, LW and LY showed Trolox equivalent antioxidant capacity (TEAC) values of 3.07, 1.87 and 1.52 mmol TE per mmol peptide, respectively. These results suggest that the MYP from sea urchin (S. nudus) gonads is a good source of antioxidant peptides with abundant tryptophan.

Characteristic antioxidant activity and comprehensive flavor compound profile of scallop (Chlamys farreri) mantle hydrolysates-ribose Maillard reaction products.

The objective of the present study was to improve the utilization of scallop (Chlamys farreri) byproducts by using Maillard reaction. Scallop mantle hydrolysates (SMHs) were prepared using neutrase then reacted with ribose. Thirty-four peptides were identified from SMHs by UPLC-Q-TOF-MS, and the abundance of Asp and Lys suggested the strong Maillard reactivity. The formation of Schiff's base as well as modification of amide I, II and III bands in Maillard reaction products (MRPs) was confirmed by ultraviolet-visible, fluorescence, and Fourier transform infrared spectroscopy. Thirty volatile compounds were produced by the reaction of SMHs with ribose. Moreover, MRPs with enhanced radical scavenging and anti-linoleic acid peroxidation activities over SMHs promoted the survival and reduced the DNA damage of HepG2 cells treated with hydrogen peroxide. These results suggest that SMHs-ribose MRPs can be potentially used as food antioxidant for suppressing of lipid oxidation or protecting of cell from oxidative damage.

Characterization of proteolysis in muscle tissues of sea cucumber Stichopus japonicus.

The proteolysis in muscle tissues of sea cucumber Stichopus japonicus (sjMTs) was characterized. The proteins from sjMTs were primarily myosin heavy chains (MHCs), paramyosin (Pm), and actin (Ac) having a molecular mass of approximately 200, 98, and 42 kDa, respectively. Based on SDS-PAGE analysis and quantification of trichloroacetic acid (TCA)-soluble peptides released, degradation of muscle proteins from sjMTs was favorable at pH 5 and 50°C. Proteolysis of MHCs was mostly inhibited by cysteine protease inhibitors, including trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane (E-64) and antipain (AP). E-64 and AP completely inhibited the degradation of Pm and Ac, while iodoacetic acid showed a partially inhibitory effect. These results indicated that the proteolysis of sjMTs was mainly attributed to cysteine proteases. Avoidance of setting the tissues at 40-50°C and slightly acidic condition and inhibition of cysteine proteases are helpful for decreasing sea cucumber autolysis.