| Red color change can be seen in all crustacea aquatic products after cooking,but the degree of red color change varies with species.Red color change of Procambarus clarkii is relatively significant.Degree of red color change is an important index to measure commodity value of crustacea aquatic products and it influences the desire of purchase and eating of consumers to a large extent.With respect to the essence of red color change of crustacea aquatic products after heating,there’s an agreement in the research field that pigment protein degenerate upon heating to release astaxanthin that can bond with pigment proteins,thus making the astaxanthin present the original red color.However,the relationship between the significant degree of red color change induced by carapace heating of P.clarkii and structures and properties of the pigment proteins still remains unknown yet.To address this problem,the red color-related pigment proteins were extracted from the carapace of P.clarkii through protein separation technology in this study.The subunit amino acid sequences were acquired through homology-based cloning and their spatial structures were analyzed by bioinformatics analysis.Influences of boiling conditions on structure and color of pigment proteins were investigated thoroughly.Based on the molecular dynamics simulation technology,influences of heating treatment on microstructure of pigment proteins were analyzed.Finally,influences of boiling conditions on red color change of carapace were discussed according to properties of pigment proteins to verify the role of pigment proteins in red color change of shrimp after heating and disclose their internal relations.Research conclusions can provide some theoretical references for heating-induced red enhancement of other crustacea aquatic products.Major research contents and results are summarized as follows:1.By analyzing the color changes of tissues like exoskeleton,epidermis and muscles before and after heating,the cephalopthorax exoskeleton was determined as the significant position of heating-induced red color change of P.clarkii.Two red color-related pigment proteins,170 k Da and 43 k Da,were isolated and purified from cephalopthorax exoskeleton by using ammonium sulfate fractional precipitation and gel filtration chromatography,which were both composed of single 21 k Da subunit,respectively.According to LC-MS/MS analysis,this 21 k Da subunit belongs to the crustacyanin family and it was named P.clarkii crustacyanin A2(Pc CRA2).The prosthetic group of pigment proteins were both determined astaxanthin through UV-vis and UPLC-MS.According to research results,the two red pigment proteins were astaxanthin binding proteins of homologous octamer and homodimer,respectively,named as Pc ABP170 and Pc ABP43.2.The gene coding sequence of Pc CRA2(cra2)was gained through homology-based cloning and its expression modes in 9 types of tissues of P.clarkii were detected through semi-quantitative and quantitative real-time fluorescence PCR(RT-PCR).Heterologous recombinant expression of Pc CRA2 was carried out by using Escherichia coil.BL21(DE3)as the host.Results demonstrated that c DNA of cra2 was 573 bp long and it encoded 190amino acids.According to the bioinformatics analysis,Pc CRA2 has“β-barrel”structure and the sole tryptophan is at the bottom of the barrel.Astaxanthin locates in the center of the“β-barrel”structure.Seven key amino acid residues likeβ-ionone ring and Gln48 form non-covalent binding.According to the RT-PCR results,cra2 is expressed in all testing tissues and its expression in surface skin is the highest.The p ET28a-cra2 prokaryotic expression vector was built and expressions were induced in DE3.The optimal induction expression conditions were adding 0.5 mmol/L IPTG at 4 h after the inoculation and inducing for 6 h under 30℃.The UV-vis results demonstrated that recombinant proteins can form specific binding with astaxanthin.3.Influences of heating temperature,heating time and Na Cl concentration on structure and color of pigment proteins were studied by circular dichroism,fluorescence spectra and UV-Vis spectra.Results demonstrated that:(1)From 60～100℃,the L~*,a~*and b~*values of pigment protein increase significantly with the increase of temperature(P<0.05).Theα-helix content declined gradually,while theβ-fold content increased gradually.The endogenous fluorescence developed red shift gradually,the UV spectra developed blue shift gradually,and the maximum visible absorption peak moved from 615 nm to 455 nm.This indicated that astaxanthin was dissociated from the unfolding“β-barrel”structure gradually.(2)As the heating time prolongs,the L~*,a~*and b~*values of pigment protein increased firstly and then decreased,reaching the peaks at 10 min.Theα-helix content decreased firstly and then became stable,while theβ-fold content increased firstly and then became stable.The endogenous fluorescence developed red shift firstly and then blue shift,indicating that structures of the pigment proteins were unfolded gradually and aggregates were formed.The maximum visible peak developed red shift from 458 nm to 462 nm,indicating that the dissociation degree of astaxanthin was increasing gradually.(3)With the increase of Na Cl concentration,the L~*value of pigment protein increased firstly and then decreased,while the a~*value was stable firstly and then declined.Solubility decreased significantly and turbidity increased greatly,accompanied with gradual reduction of the SDS-PAGE band corresponding to subunit.This indicates that Na Cl could promote thermal aggregation of pigment proteins.4.Microstructure and binding force changes of pigment proteins at 298 K,373K and473 K could be studied by molecular dynamics simulation,MM-GBSA calculation and decomposition binding free energy.Results showed that in 100 ns of simulation,the fluctuation values of root mean square deviation(RMSD)and root-mean-square fluctuation(RMSF)of pigment protein increased significantly with the increase of temperature,accompanied with great fluctuation degree.The radius of gyration increased to some extent,peptide chain extended,and hydrophobic groups were exposed in solvent,thus increasing the accessible surface area of solvent from 51 nm~2to 56 nm~2and decreasing hydrogen bonds from 135 to about 125.Theα-helix in the secondary structure was destroyed and the tertiary structure content decreased by 25%.Due to opening loop in the Asp94-Ser101 zone,astaxanthin was separated from the complex.The average free binding energy of complex always increases with the increase of temperature.This reveals that the binding between proteins and astaxanthin is weakened as temperature rises,and the trend for astaxanthin disassociation from the complex is increased.5.Effects of cooking temperature,cooking time and saline concentration on red color change of P.clarkii were investigated by single-factor and response surface experiment.Results showed that the brightness(L~*)and redness(a~*)of the whole shrimp increased significantly with the increase of temperature.The L~*value of the whole shrimp increased significantly and then changed slightly,while a~*value presented an inverted V-shaped variation with the cooking time.The L~*value of the whole shrimp presented an inverted V-shaped variation with the increase of saline content,while the a~*value was stable firstly,then increased and finally declined.In a word,the variation trends of L~*value and a~*value of the whole shrimp with cooking temperature,cooking time and saline concentration are basically corresponding with pigment protein Pc ABP170.According to the response surface results,cooking temperature is the primary influencing factor of a~*value,followed by saline concentration and cooking time successively. |
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