Solubilization of fish myofibrillar proteins in NaCl and KCl solutions: A DIA-based proteomics analysis.

Understanding the basic solubilization of fish myofibrillar proteins (MPs) in common monovalent chloride solutions is crucial for muscle food processing. In this study, the differential proteomic profiles of MPs during extraction and solubilization in NaCl and KCl solutions were investigated by using advanced four-dimensional data-independent acquisition (4D DIA) quantitative proteomics for the first time. Compared to routine biochemical analysis, this could provide insights into the solubilization of muscle proteins. We ensure the consistency of the effective ionic strength of NaCl and KCl buffers by adjusting the conductivity. The results showed that NaCl extractor mainly facilitated the solubilization of cytoskeletal proteins, biochemical enzymes, and stromal proteins compared to KCl, such as tubulin, myosin-9, collagen, plectin, protein phosphatase, and cathepsin D. However, no significant difference was observed in the extraction of major sarcomeric proteins, including myosin, actin, troponin C, myosin-binding protein C, M-Protein, α-actinin-3, and tropomyosin.

Unraveling the binding mechanisms of transglutaminase and substrate subjected to microwaves: Molecular docking and molecular dynamic simulations.

Previous studies showed that transglutaminase (TGase) and microwaves acted synergistically to improve the functional properties of proteins. The mechanism behind this has yet to be elucidated. In this study, the phenomenon of microwaves enhancing TGase activity was experimentally validated. Molecular docking and molecular dynamics simulations revealed that moderate microwaves (105 and 108 V/m) increased the structural flexibility of TGase and promoted the orientation of the side chain carboxylate anion group on Asp255, driving the reaction forward. Also, TGase underwent partial transformation from α-helix to turns or coils at 105 and 108 V/m, exposing more residues in the active site and facilitating the binding of the substrate (CBZ-Gln-Gly) to TGase. However, 109 V/m microwaves completely destroyed the TGase structure, inactivating the enzyme. This study provides insights into the molecular mechanisms underlying the interactions between TGase and substrate subjected to microwaves, promoting the future applications of TGase and microwaves in food processing.

Postmortem Muscle Proteome Characteristics of Silver Carp (Hypophthalmichthys molitrix): Insights from Full-Length Transcriptome and Deep 4D Label-Free Proteomic.

The coverage of the protein database directly determines the results of shotgun proteomics. In this study, PacBio single-molecule real-time sequencing technology was performed on postmortem silver carp muscle transcripts. A total of 42.43 Gb clean data, 35,834 nonredundant transcripts, and 15,413 unigenes were obtained. In total, 99.32% of the unigenes were successfully annotated and assigned specific functions. PacBio long-read isoform sequencing (Iso-Seq) analysis can provide more accurate protein information with a higher proportion of complete coding sequences and longer lengths. Subsequently, 2671 proteins were identified in deep 4D proteomics informed by a full-length transcriptomics technique, which has been shown to improve the identification of low-abundance muscle proteins and potential protein isoforms. The feature of the sarcomeric protein profile and information on more than 30 major proteins in the white dorsal muscle of silver carp were reported here for the first time. Overall, this study provides valuable transcriptome data resources and the comprehensive muscle protein information detected to date for further study into the processing characteristic of early postmortem fish muscle, as well as a spectral library for data-independent acquisition and data processing. This batch of muscle-specific dependent acquisition data is available via PRIDE with identifier PXD043702.

Insight into Ionic Strength-Induced Solubilization of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix): Structural Changes and 4D Label-Free Proteomics Analysis.

In this study, changes in the physical, structural, and assembly characteristics of silver carp myofibrillar proteins (MPs) at different ionic strength (I) values were investigated. Moreover, the differential proteomic profile of soluble MPs was analyzed using 4D proteomics based on timsTOF Pro mass spectrometry. Solubility of MPs significantly increased at high I (>0.3), and the increase in I enhanced the apparent viscosity, fluorescence intensity, surface hydrophobicity, and α-helix content of MPs solution. Particle size and sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns also supported the solubility profiles. Transmission electron microscopy and atomic force microscopy observations revealed the morphological assembly and disassembly of MPs under different I conditions. Finally, proteomic analysis revealed the evolution law of salt-induced solubilization of MPs and the critical molecular characteristics in different I environments. The number of differentially abundant proteins (DAPs) decreased with the increase of I, and most DAPs related to the muscle filament sliding, contraction and assembly, actinin binding, and actin filament binding. The soluble abundance of myosin and some structural proteins was dependent on I, and structural proteins in the Z-disk and M-band might contribute to the solubilization of myosin. Our findings provide insightful information about the impact of common I on the solubility pattern of MPs from freshwater fish.

Microwave heating process of moderate-minced surimi based on multiphase porous media model.

Moderately processed surimi products exhibit better nutrient retention and enhanced gels, and the great potential of microwaves application and changes in the way of chopping meat has been reported by previous research. In this study, a systematic analysis of the novel surimi product was made to explore the heat and mass transfer characteristics. A porous media model combining electromagnetic heat and hygroscopic expansion was developed to evaluate this process, and its accuracy has been verified experimentally. It was found that the dielectric characterization of multiphase mixture system has great influence on the results, the complex refractive index mixture equation was used due to its lowest root-mean-square error value. In addition, the effect of moderate processing on microwave heating was examined in terms of porosity changes. However, nonuniform temperature distributions were found in the higher porous samples, especially when the porosity is greater than 0.81. Moreover, the developed model was coupled with the evaluation for gel properties and the results showed the significant effect of moderate crushing on the gel quality during the microwave heating process.

Changes in physicochemical properties of silver carp (Hypophthalmichthys molitrix) surimi during chilled storage: The roles of spoilage bacteria.

Chilled surimi has become increasingly popular owing to its superior texture and freshness. In this study, changes in the microbiota and gel properties during chilled surimi storage, and the contributions of dominant bacteria to the physicochemical properties of chilled surimi were investigated. The results showed that Pseudomonas gessardii, Aeromonas media, and Acinetobacter johnsonii were the dominant bacteria during chilled surimi storage. P. gessardii was the key bacteria that degraded protein in the process of surimi spoilage, which led to high total volatile base nitrogen (TVB-N), trichloroacetic acid (TCA)-soluble peptides as well as poor gel properties. Both P. gessardii and A. media were high putrescine producers, whereas only A. media produced cadaverine. In this study, spoilage microorganisms in chilled surimi were investigated for the first time, and it was found that P. gessardii had the greatest influence on surimi quality, which provides a research basis for in-depth study on the mechanism of microbial spoilage and the preservation of chilled surimi.

Redox Proteomic Analysis Reveals Microwave-Induced Oxidation Modifications of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix).

To provide an insight into the oxidation behavior of cysteines in myofibrillar proteins (MPs) during microwave heating (MW), a quantitative redox proteomic analysis based on the isobaric iodoacetyl tandem mass tag technology was applied in this study. MPs from silver carp muscles were subjected to MW and water bath heating (WB) with the same time-temperature profiles to eliminate the thermal differences caused by an uneven energy input. Altogether, 422 proteins were found to be differentially expressed after thermal treatments as compared to that with no heat treatment. However, MW triggered a larger number of proteins and cysteine sites for oxidation. Myosin heavy chain, myosin-binding protein C, nebulin, α-actinin-3-like, and titin were found to be highly susceptible to oxidation under microwave irradiation. Notably, MW caused such modifications at cysteine site 9 in the head of myosin, revealing the enhancement mechanism of MP gelation by excess cysteine cross-linking during microwave processing. Furthermore, Gene Ontology and functional enrichment analyses suggested that the two thermal treatments resulted in some differences in ion binding, muscle cell development, and protein-containing complex assembly. Overall, this study is the first to report the redox proteomic changes caused by MW and WB treatments, thus providing a further understanding of the microwave-induced oxidative modifications of MPs.

Inhibitory effect of microwave heating on cathepsin l-induced degradation of myofibrillar protein gel.

This work was aimed to compare the effect of microwave (MW) heating on the cathepsin L (Cat L)-induced degradation of myofibrillar protein (MP) gels with that of water bath (WB) heating. First, Cat L from silver carp was purified and determined to be 45 kDa. The gel strength of the MW-heated MP gels were significantly higher than those of the WB-heated when Cat L was added (P < 0.05). The gel electrophoresis pattern and scanning electron microscopy analysis indicated that MW heating inhibited the Cat l-induced hydrolysis of MP gels. In addition, the number of sulfhydryl groups and surface hydrophobicity of MW-heated gels were lower than those of WB-heated gels when Cat L was added. These results indicated that MW heating could effectively weaken the degradation of Cat L on MP gels by manipulating disulfide bonds and hydrophobic amino acids, resulting in good gel properties and a compact protein network.

Effect of lipase incorporation on gelling properties of catfish (Clarias lazera) surimi and its mechanism.

BACKGROUND: Recently, fatty fish have been utilized as a potential approach for the fabrication of surimi products, with the yield of fatty fish surimi being > 10 000 tons in 2019. However, the gelling properties of catfish surimi can be influenced by intermuscular lipid. Lipase could effectively enhance the gel quality of catfish surimi gels, although the chemical forces involved in gel formation and alteration in lipid and protein oxidation status are not well understood. The present study investigated the gelation-enhancing effects of lipase on catfish surimi based on changes in chemical oxidation interactions. RESULTS: The addition of 7.5 g kg-1 lipase significantly increased the hydrophobic interactions and disulfide bond contents, both of which facilitated gel formation, in surimi gels. The 2-thiobarbituric acid reactive substance and carbonyl concentrations demonstrated that lipase promoted lipid and protein oxidations. Furthermore, an appropriate dose of malondialdehyde accelerated protein oxidation, thereby resulting in the covalent cross-linking of proteins. Consequently, the gel strength increased from 55.72 to 127.71 g × cm with lipase contents of up to 7.5 g kg-1 , and strong chemical cross-linking and a compact network were observed via sodium dodecyl sulfate polyacrylamide gel electrophoresis and scanning electron microscopy. However, excessive oxidation led to the degeneration of the gel matrix. A schematic mechanism, mainly based on the chemical changes, is proposed. CONCLUSION: The present study revealed the gelation mechanism of catfish surimi gels with lipase, and suggested that lipase treatments may be an effective approach for improving the textural properties of fatty fish surimi gels. © 2021 Society of Chemical Industry.

Catalytic effect of transglutaminase mediated by myofibrillar protein crosslinking under microwave irradiation.

Microwave (MW) heating improved the activity of transglutaminase (TGase) by inducing conformational changes due to structural modification. However, when TGase and myofibrillar protein were heated, the solubility and degree of crosslinking were similar. Further, the gel properties of the mixed solution pre-gelled by MW heating were lower than that obtained with water bath (WB) pre-gelling. We compared the effects on myofibrillar proteins at the same heating rate, our results showed that MW promoted aggregation, as the particle distribution tended toward larger molecular size. The increase of random coil as investigated by circular dichroism (CD) indicated that WB induced the unfolding of myofibrillar protein. MW enhanced intermolecular forces by engendering more disulfide bonds, which hindered the catalysis by TGase. Finally, SDS-PAGE indicated that the myosin molecules had more head crosslinking during MW treatment. MW and WB cause different response behaviors of myofibrillar protein, thereby affecting the catalytic effect of TGase.

Intervention of transglutaminase in surimi gel under microwave irradiation.

Transglutaminase (TGase) was selected as model enzyme to investigate the effects of microwave (MW) heating on its activity and structure compared to water bath (WB) heating. MW heating can enhance the activity of TGase and reach the maximum at 20 min, whereas conduction heating has little effect on the activity of TGase. The difference of dielectric properties between MW heating and WB heating were not obvious, but MW heating had higher conductivity than WB heating. The results of ultraviolet and fluorescence spectra show that MW heating can change the enzyme activity by changing the conformation of TGase. The decrease of α-helix and an increase of ß-sheet and ß-turn investigated by circular dichroism (CD) indicated the secondary structures of TGase were changed when treated by MW heating. Further gel properties test confirmed that TGase treated by MW could improve the functional and mechanical properties of surimi gel.

Protein structural development of threadfin bream ( Nemipterus spp.) surimi gels induced by glucose oxidase.

This study investigated the effect of glucose oxidase on the gel properties of threadfin bream surimi. The gel strength of surimi increased with the addition of 0.5‰ glucose oxidase after two-step heating. Based on the results of the chemical interactions, the hydrophobic interaction and disulfide bond of glucose oxidase-treated surimi samples increased compared with the control samples at the gelation temperature and gel modori temperature. The surface hydrophobicity of samples with glucose oxidase and glucose increased significantly ( p < 0.05) and total sulfhydryl groups decreased significantly ( p < 0.05). The analysis of Raman spectroscopy shows that the addition of glucose oxidase induced more α-helixes to turn into a more elongated random and flocculent structure. Glucose oxidase changes the secondary structure of the surimi protein, making more proteins depolarize and stretch and causing actomyosin to accumulate to each other, resulting in the formation of surimi gel.