| The"blue granary"strategy built around aquatic products has optimized the food supply for Chinese residents and ensured their dietary health.Culter alburnus,known for its delicate texture and high economic value,is a famous freshwater fish in Hubei Province.C.alburnus,highly sensitive to stress,has poor oxygen tolerance,and is prone to mortality after being caught or released from the water.Thus,it is usually transported and sold through frozen storage or cold preservation.Myofibrillar protein（Mfp）,the main component of fish protein（60%-70%）,is prone to denaturation and deterioration of gel performance during frozen storage,which is the key factor leading to the decline of fish quality.Currently,sucrose or sorbitol are commercially used as cryoprotectants to inhibit the deterioration of fish protein during frozen storage,with unnecessary calories and sweetness added to the fish.Meanwhile,functional oligosaccharides not only have the characteristics of low sweetness and low calories,but also have the potential to inhibit the deterioration of fish protein because of their strong hydroxyl properties.As a natural macromolecule,egg white protein（EWP）has balanced amino acids and a low price,which has a good effect on improving the gel properties of fish protein products.Therefore,three types of functional oligosaccharides,including isomalto-oligosaccharide（IMO）,carrageenan oligosaccharide（CGO）,and xylo-oligosaccharide（XO）,were selected.Oligosaccharides mixtures were subsequently prepared with EWP.In addition,oligosaccharides were glycosylated with EWP to improve the molecular structure and functional properties of Mfp during frozen storage.Firstly,the influence of oxidation on the intermolecular interactions of C.alburnus Mfp was investigated.Additionally,the synergistic effect of the mixture of oligosaccharides and EWP on improving the functional properties of C.alburnus Mfp during frozen storage was analyzed,Moreover,the mixture of oligosaccharide and EWP was glycosylated to explore the effects of the glycosylation complex on the oxidative denaturation of C.alburnus Mfp and to clarify the regulation mechanism of the glycosylation complex on the molecular structure and gel properties of C.alburnus Mfp during frozen storage.Meanwhile,the micro-mechanism and macro laws of improving C.alburnus muscle quality by glycosylation complex under the environmental temperature fluctuation during simulated processing and consumption scenarios were investigated.The main research content and results are as follows:1.The changes in the molecular interactions of C.alburnus myofibrillar protein（Mfp）during oxidative deterioration was explored,as well as the effects of different mixtures（mix）of oligosaccharide and EWP（IMO/EWO-mix,CGO/EWP-mix,and XO/EWP-mix）on Mfp oxidation during multiple freeze-thaw cycles were investigated.The results showed that oxidation caused Mfp conformational unfolding,and exposure of internal hydrophobic groups.It resulted in decreased hydrogen and ionic bond content while increasing hydrophobic interactions and disulfide bond content in Mfp heat-induced gels.During multiple freeze-thaw cycles,the mixture of oligosaccharide and EWP reduced the oxidation of Mfp side chains and unfolding of conformational sites and inhibited the decrease in protein solubility.Moreover,the molecular docking results indicated that oligosaccharides docked with Mfp domains via hydrogen bonding rather than macromolecule EWP.It suggested the significant role of oligosaccharides in delaying changes in Mfp molecular structure.After four freeze-thaw cycles,compared with the blank group,the gel strength of Mfp added with IMO/EWO-mix,CGO/EWP mix and XO EWP-mix increased by 38.54%,27.35%and 65.66%,respectively,and the water holding capacity of Mfp gel increased by15.84%,39.82%and 14.40%,respectively.It shows that the mixture of oligosaccharide and EWP effectively improved the network strength of Mfp gel and delayed the deterioration rate of gel performance.2.Using IMO as a representative of oligosaccharides,the mixture of oligosaccharides and EWP（IMO/EWP-mix）was added to Mfp to analyze its influence on the structural and functional properties of Mfp molecules during frozen storage.The results showed that,at the beginning of frozen storage,adding EWP or IMO/EWP-mix had a higher gel elasticity and a shorter T2 relaxation time of the Mfp gel compared with the blank group.It indicated that the EWP contained in the IMO/EWP-mix could enhance the strength of the Mfp gel network by promoting cross-linking and filling effects.During frozen storage,the addition of IMO and IMO/EWP-mix significantly suppressed the generation of carbonyl and dityrosine groups in the side chains of Mfp,inhibited the exposure of internal hydrophobic residues,and the decrease inα-helical content.It suggested that the oligosaccharides contained in the IMO/EWP-mix could enhance the stability of Mfp molecular structure during frozen storage.After 60 days of frozen storage,compared with the blank group,the emulsification activity and emulsification stability of Mfp samples with the addition of IMO/EWP-mix increased by 21.85%and 32.29%,respectively.In addition,the ionic and hydrogen bond strength of the gel increased by 28.47%and17.81%,respectively.It indicates that the IMO/EWP-mix could slow down the degradation rate of the interfacial properties of Mfp during frozen storage and maintain its intermolecular bonding interactions.Compared with the blank group,the gel strength of Mfp increased by 16.43%and 38.07%with the addition of EWP and IMO,respectively,with the addition of IMO/EWP-mix,the strength increased by55.83%,indicating a synergistic effect of oligosaccharides and EWP in improving the gel properties of Mfp during frozen storage.3.The influence and regularity of oligosaccharides and EWP glycosylation complexes on the oxidation-induced denaturation of C.alburnus Mfp were investigated.Analysis of the grafting ratio,browning products,and protein oxidation products indicated that IMO and XO showed better glycosylation reactivity and safety.Compared to the IMO/EWP-mix and XO/EWP-mix,the Fe3+reduction capacity of IMO-EWP-gly and XO-EWP-gly increased by 18.93%and 18.87%after 9 hours of reaction,while the ABTS+and DPPH·free radical scavenging capacity increased by60.17%,14.35%and 32.87%,15.86%,respectively.The Mfp samples added with IMO-EWP-gly and XO-EWP-gly,the free amino acid content,Ca2+-ATPase activity,and internal fluorescence intensity higher than IMO/EWP-mix and XO/EWP-mix,with theα-helix content increased from 29.5%and 30.2%to 36.1%and 37.5%,respectively.It indicated that glycosylation complexes inhibited the oxidation of side chain groups and changes of spatial conformational of Mfp caused by free radicals.Meanwhile,the average particle size of IMO-EWP-gly and XO-EWP-gly decreased by 61.55%and 56.76%,respectively,and the active sulfhydryl groups increased by14.62%and 27.56%.It showed glycosylation complexes have better crosslinking-promoting and filling effects than the IMO/EWP-mix and XO/EWP-mix.Moreover,the gel strength of non-oxidized Mfp samples added with IMO-EWP-gly and XO-EWP-gly increased by 19.82%and 37.58%,respectively;while after oxidation,the gel strength of Mfp increased by 27.45%and 28.69%,respectively.It indicated that glycosylation complexes more effectively reduce the structural changes of Mfp molecules caused by free radical oxidation,and improve the gel network strength of Mfp,thereby delaying the deterioration rate of Mfp gel properties.4.The regulatory effect and mechanism of glycosylation complexes on the structure and gel properties of Mfp molecules in frozen storage were investigated.The results showed that the XO-EWP-gly could enhance the low-temperature stability of the Mfp molecule structure in frozen storage,reduce the oxidation of Mfp sulfhydryl,free amino groups,inhibit exposure to internal hydrophobic residues,and decline irregular aggregation.After 60 days of frozen storage,the protein solubility,emulsification ability and emulsification stability of Mfp samples added with XO-EWP-gly improved by 29.95%,26.55%,and 17.55%,respectively.Meanwhile,the content of ionic and hydrogen bonds in the gel increased by 43.14%and 24.83%,respectively.It indicated that the XO-EWP-gly could more effectively protect the integrity of the spatial conformation of Mfp molecules in frozen storage,delay the deterioration rate of the Mfp interfacial properties,and maintain the stability of intermolecular interactions than the blank group.In addition,compared with the XO/EWP-mix,the gel strength of Mfp gel added with the XO-EWP-gly increased by16.87%.Moreover,rheological properties and SEM microstructure analysis showed that the Mfp gel added with XO-EWP-gly has a higher storage modulus,polymerization degree,and a denser gel network structure.This revealed that EWP is more conducive to improving the gel properties of Mfp in frozen storage after glycosylation.5.The mechanism of glycosylation complexes on the quality of C.alburnus muscle product under the processing-consumption scenario and environment fluctuations was investigated.The results showed that after two freeze-thaw cycles,adding XO/EWP-mix and XO-EWP-gly could significantly improve the elasticity,cohesiveness,and chewiness of C.alburnus surimi gel compared with XO and EWP.It implied a synergistic effect of oligosaccharides and EWP in improving the textural properties of C.alburnus muscle products.Moreover,XO-EWP-gly could better delay the decrease of hydrogen and ionic bonds in fish mince gel,maintain intermolecular interactions,and prevent the decrease of the water-holding capacity of fish muscle products.Meanwhile,After two freeze-thaw cycles,compared with XO/EWP-mix,XO-EWP-gly reduced the TCA-soluble peptide content and TBARS value in fish mince by 17.83%and 19.25%,respectively.After four freeze-thaw cycles,the breaking force and strength value of C.alburnus surimi gel with added XO-EWP-gly increased significantly by 31.71%and 46.13%,respectively.The results showed that glycosylated complexes had the better free radical scavenging ability,slowing down the oxidative degradation of lipids and proteins,inhibiting the production of unpleasant flavor substances in C.alburnus muscle products,and improving the strength of the C.alburnus muscle gel network.In summary,the study demonstrated a synergistic effect of mixtures and glycosylation complexes of oligosaccharides and EWP on regulating the molecular structure and gel properties of Mfp in C.alburnus muscle during frozen storage.The glycosylation complexes of the oligosaccharides and EWP are more significant improvements in the gel properties of Mfp and the quality of C.alburnus muscle products during frozen storage.The results provide a theoretical foundation and reference for the application in developing new cryoprotective agents. |
