Functional Properties Of Gonad Proteins And Hydrolysates From Sea Urchin

Sea urchin is one of the important costly sea food,and gonad is the edible part of a sea urchin.In the coast of Huanghai Sea and Bohai Sea of China,Strongylocentrotus nudus?S.nudus?,Glyptocidaris crenularis?G.crenularis?and Strongylocentrotus intermedius?S.intermedius?are important aquatic economic animals.However,studies on functional properties of gonad protein derived from these three species of sea urchins have not been reported so far.As regard to these three species of sea urchins,functional properties of sea urchin gonad protein isolates were studied and compared in the present study.Based on these results,proteomics was used to detect mechanism of condition factor and fatty acid content varation of S.nudus during the fishing season.Otherwise,Fe²⁺chelating property and antioxidant activity of S.nudus gonad hydrolysates were further detected.In this study,S.nudus,G.crenularis and S.intermedius gonad protein isolates?snGPIs,gcGPIs and siGPIs?were extracted by pH shift method,and their functional properties were determined and compared with those of defatted gonads?snDGs,gcDGs and siDGs?.The results demonstrated that protein content and water-and oil-holding capacities of the GPIs were significantly enhanced compared with those of DGs?p<0.05?.Moreover,the GPIs had better foaming property and foaming stability than that of the DGs.In all samples,gcGPIs had higher emulsifying activity indexes from pH 6 to 8?p<0.05?.Furthermore,the GPIs had lower?-helix and?-turn contents and a higher?-sheet content compared with those of DGs.These results suggest that the GPIs of three species of sea urchin can offer new product opportunities as raw material.To investigate the variation in condition factor of S.nudus,the gonads were collected in May?MAY?,June?JUN?,July?JUL?,at the beginning?AUG-b?and end of August?AUG-e?.Gonadal proteins were identified by isobaric tagging for relative and absolute quantitation?iTRAQ?,and the 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 gonad index?GI?and protein content.These included 6-phosphogluconate dehydrogenase,decarboxylating isoform X2?6PGD?,CAD protein,myoferlin isoform X8,ribosomal protein L36?RL36?,isocitrate dehydrogenase?mitochondrial isoform X2??IDH?,multifunctional protein ADE2 isoform X3,sperm-activating peptides?SAPs?and aldehyde dehydrogenase?mitochondrial??ALDH?.However,TGFBI had no correlation with 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 the changes of S.nudus gonad condition factor.iTRAQ-based proteomics analysis was used to detect the variation of fatty acid content in the gonads of S.nudus which were collected in different fishing season.A total of 7 fatty acid synthetic related DEPs were screened out,including arylsulfatase?Ars?,glutamine synthetase?GS?,thimet oligopeptidase-like?TOP?,dipeptidyl peptidase 2?DPP 2?,sperm phosphodiesterase 5?sperm PDE 5?,retinoid-inducible serine carboxypeptidase and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 isoform X2.Quantitative real time PCR?qRT-PCR?analysis indicated that the upregulation of mRNA relative expression level of Ars,TOP and sperm PDE 5 in AUG-b/AUG-e-MAY,JUL/AUG-e-MAY and AUG-b-JUN groups,and the protein level of these DEPs was also enhanced.Despite the increasment of mRNA relative expression level of GS and DPP 2 in AUG-b/AUG-e-MAY and AUG-e-MAY groups,the protein level of these DEPs decreased in these groups.Ars,GS,TOP and DPP 2 can be used as biomarkers for content variation of stearic acid,oleic acid,C18:2n6c,arachidic acid and arachidonic acid in AUG-e-MAY group.Futhermore,Ars and GS may act as biomarkers of palmitic acid,C17:1,C18:1n9t,arachidic acid and?-linolenic acid in AUG-b-MAY group.S.nudus defatted gonads were hydrolyzed by four enzymes,including trypsin,papain,alcalase and neutrase to release Fe²⁺chelating peptides.Sea urchin gonad hydrolysates?SGHs?released by alcalase showed the highest Fe²⁺chelating activity with EC₅₀ of 0.27±0.01 mg/mL.Amino acid analysis indicated that SGHs was lack of Cys,and Met was not detected in sea urchin gonad hydrolysates-Fe²⁺(SGHs-Fe²⁺)complexes.Ultraviolet-visible and fourier-transform infrared spectroscopy spectra revealed that Fe²⁺could bind to the SGHs by Ser,Gly,His and so on.Sequencing result of the SGHs was detected by an UPLC-Q-TOF-MS/MS,and reaction heat change of three peptides binding with Fe²⁺was further investigated,including Glu-Ser-Val-Asn-Arg-Tyr-Phe?ESVNRYF?,Ser-Arg-Leu-Val-Asn-Pro-Asn?SRLVNPN?and Ser-Ser-Arg-Leu-Val-Asn-Pro-Asn?SSRLVNPN?.Results indicated that these peptides bind Fe²⁺with a weak binding affinity,and endothermic chelate reactions are drove by entropy.These results suggest that SGHs is a good candidate for preparing SGHs-Fe²⁺complexes.In silico assessment was used to predict and verified S.nudus gonad major yolk protein?MYP?derived antioxidant peptides.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 obtained a score over 0.5 by PeptideRanker.The peptides LW,TW and LWK showed stronger antioxidant capacity with IC₅₀ values of 8.85,9.59 and 9.62 mmol/L,respectively,compared to that of glutathione?10.81 mmol/L?.Furthermore,AW,LW and LY showed Trolox equivalent antioxidant capacity?TEAC?values of 3.07,1.87 and 1.52 mmoL TE/mmoL peptide,respectively.These results suggest MYP from S.nudus gonads is a good source of antioxidant peptides with abundant tryptophan.In summary,S.nudus and G.crenularis are sources of gonad protein isolates with good functional properties.Condition factor variation of S.nudus gonad has been revealed by 10DEPs which have correlation with GI and protein content during the fishing season.Furthermore,4 fatty acid synthesis ralated DEPs are biomarkers of fatty acid content variation in S.nudus gonad during the fishing season.The alkaline protease hydrolysate of S.nudus gonad has good Fe²⁺chelating activity.Moreover,antioxidant peptides derived from S.nudus MYP has good antioxidant property.Therefore,gonad protein of S.nudus,G.crenularis and S.intermedius which distribute in the Huanghai Sea and the Bohai Sea has good functional properties and potential application value.