The proteomic evidence on protein oxidation in pea protein concentrate-based low-moisture extrudates and its inhibition by antioxidants derived from plant extracts.

This study aimed to assess the antioxidant activity of golden chlorella (GoC) and grape pomace (GrP) extracts both in vitro and in pea protein-based extrudates. We hypothesized that GoC/GrP would limit oxidation of proteins in the extrudates compared with commercial antioxidants. The results showed that GoC extract was effective in metal chelation and GrP extract possessed excellent radical scavenging activity and reducing power. Protein oxidation inevitably occurred after low-moisture extrusion in terms of elevated level of protein carbonyls and the gradual loss of thiols. LC-MS/MS revealed that the monoxidation and 4-hydroxynonenal adduction were the major oxidative modifications, and legumin was the most susceptible globulin for oxidation. The GoC/GrP extracts effectively retarded the oxidation progress in extrudates by lower intensity of oxidized peptides, whereas protein electrophoretic profiles remained unaffected. This study highlighted the great potential of GoC/GrP as natural antioxidants in plant-based foods.

Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

Oxidation and side-chain modifications decrease gastrointestinal digestibility and transport of proteins from salted bighead carp fillets after frozen storage.

This study examined the effects of protein oxidation on digestion behavior. The oxidation levels and in vitro digestibility of myofibrillar proteins from fresh-brined and frozen bighead carp fillets were investigated, and the intestinal transport property was characterized by comparing the peptides on both sides of the intestinal membrane. Frozen fillets showed high oxidation levels, low amino acid content and in vitro protein digestibility, which were further increased by brining. After storage, the number of modified peptides from myosin heavy chain (MHC) increased over 10-fold in NaCl-treated samples (2.0 M). Various types of side-chain modifications in amino acids were identified, such as di-oxidation, α-aminoadipic semialdehyde (AAS), γ-glutamic semialdehyde (GGS), and protein-malondialdehyde (MDA) adducts, mainly originating from MHC. The Lysine/Arginine-MDA adducts, AAS, and GGS decreased protein digestibility and their intestinal transportation. These findings suggest that oxidation impacts protein digestion and should be considered in food processing and preservation strategies.

Decolorization of porcine hemoglobin hydrolysates: The role of peptide characteristics and pH values.

The unpleasant color caused by heme limits the utilization of hemoglobin as a food ingredient. Enzymatic hydrolysis has been used to decolorize hemoglobin, but the underlying mechanisms are poorly understood. The aim of this study was to investigate the decolorization efficiency of porcine hemoglobin using different enzymes and final pH values, and to elucidate their influence on decolorization. Based on higher yields and better decolorization, hemoglobin hydrolysates produced by papain, bromelain, savinase, and protease A were further studied. Compared to hydrolysates by savinase and protease A, a higher proportion of histidine-containing peptides was responsible for better decolorization by papain and bromelain. For all hydrolysates, a moderate reduction in pH to 4.0-5.0 facilitated decolorization of the hydrolysates. Similar peptide profiles of hydrolysates from the same enzyme treatment reflected that pH mainly affected the precipitation of the heme-containing fraction through heme-heme interaction rather than heme-peptide interaction. Overall, this study sheds light on the use of enzymatic hydrolysis to remove the heme group from hemoglobin. PRACTICAL APPLICATION: Slaughterhouses produce tons of protein-rich blood each year. Due to the presence of the heme group in hemoglobin, blood has a dark red color and metallic taste, making it generally unacceptable for consumers. This study provided information on the decolorization of porcine hemoglobin by removing the heme fraction, which should facilitate the utilization of decolored hemoglobin hydrolysates as nutritional food ingredients.

Uncovering quality changes of salted bighead carp fillets during frozen storage: The potential role of time-dependent protein denaturation and oxidation.

This study aimed to find the specific relationship between quality traits and myofibrillar proteins (MPs) alteration of salted fish during frozen storage. Protein denaturation and oxidation occurred in frozen fillets, with the denaturation occurring before oxidation. In the pre-phase of storage (0-12 weeks), protein structural changes (secondary structure and surface hydrophobicity) were closely related to the water-holding capacity (WHC) and textural properties of fillets. The MPs oxidation (sulfhydryl loss, carbonyl and Schiff base formation) were dominated and associated with changes in pH, color, WHC, and textural properties during the later stage of frozen storage (12-24 weeks). Besides, the brining at 0.5 M improved the WHC of fillets with less undesirable changes in MPs and quality traits compared to other concentrations. The 12 weeks was an advisable storage time for salted frozen fish and our results might provide an available suggestion for fish preservation in aquatic industry.

Physicochemical properties, texture, and in vitro protein digestibility in high-moisture extrudate with different oil/water ratio.

Oil addition is challenging during high-moisture extrusion due to the negative fiber formation effects. A previous study found that oil-in-water (O/W) emulsions could significantly increase the oil content in high-moisture extrudates, but the molecular mechanism remained unclear. This study aimed to determine O/W emulsion influence on protein physicochemical properties in SPI extrudates during high-moisture extrusion. O/W emulsions were mixed with soy protein isolates (SPI) to prepare extrudates with oil/water ratios of 0/65, 4/61, and 8/57 (w/w). SDS-PAGE and ATR-FTIR analysis showed that higher oil/water ratios enhanced protein aggregation and promoted alteration from ß-sheet to random coil in SPI extrudates, which could be correlated to the reduction of protein solubility. The color was altered to lighter and yellow, and hardness, chewiness, and fiber degree decreased with increased oil/water ratios in SPI extrudates. In addition, in vitro digestion analyses showed that higher oil content contributed to improved protein digestibility.

Protein phosphorylation profile of Atlantic cod (Gadus morhua) in response to pre-slaughter pumping stress and postmortem time.

This study aimed to evaluate the effect of pumping stress (pumping and pumping-resting) and postmortem time (before and after rigor mortis) on phosphorylation profiles of myofibrillar protein (MP) and sarcoplasmic protein (SP) of Atlantic cod (Gadus morhua) fillets. The result showed that MP had higher global phosphorylation levels than SP regardless of stress condition and postmortem time. The pumping process resulted in significant changes in phosphorylation of structural proteins including myosin heavy and light chains. Pumping also affected the phosphorylation status of heat shock proteins and metabolic enzymes involved in the glycolytic pathways, indicating the possible role of phosphorylation in regulating energy hemostasis of fish under stressful conditions. The pumping-induced phosphorylation changes mainly occurred before rigor mortis, and postmortem time affected the phosphorylation status to a less extent. This work contributes to a deeper understanding on protein phosphorylation affected by pre-slaughter stress and postmortem time of fish.

Increase of Kokumi γ-Glutamyl Peptides in Porcine Hemoglobin Hydrolysate Using Bacterial γ-Glutamyltransferase.

The kokumi sensation of protein hydrolysates could be enhanced by γ-glutamylation through forming a series of γ-glutamyl di- and tri-peptides. In this study, porcine hemoglobin hydrolysate was γ-glutamylated using enzymes from Bacillus amyloliquefaciens (Ba) or Bacillus licheniformis (Bl), which are sold as glutaminases but identified as γ-glutamyltransferases (GGTs). To yield more γ-glutamyl peptides, reaction conditions were optimized in terms of GGT source (BaGGT and BlGGT), substrate concentration (10, 20, and 40%), reaction time (3, 6, 12, and 24 h), and glutamine supplementation (20, 40, and 80 mM). Results showed that both the GGTs had the highest transpeptidase activity at similar pH values but different temperatures. In addition, BaGGT had stronger catalytic ability to form γ-glutamyl dipeptides, while BlGGT was more capable to generate γ-Glu-Val-Gly. Adding glutamine was more efficient to obtain more target peptides than adjusting the hydrolysate concentration and reaction time. This study contributes to the valorization of animal side streams.

Recent advances on characterization of protein oxidation in aquatic products: A comprehensive review.

In addition to microbial spoilage and lipid peroxidation, protein oxidation is increasingly recognized as a major cause for quality deterioration of muscle-based foods. Although protein oxidation in muscle-based foods has attracted tremendous interest in the past decade, specific oxidative pathways and underlying mechanisms of protein oxidation in aquatic products remain largely unexplored. The present review covers the aspects of the origin and site-specific nature of protein oxidation, progress on the characterization of protein oxidation, oxidized proteins in aquatic products, and impact of protein oxidation on protein functionalities. Compared to meat protein oxidation, aquatic proteins demonstrate a less extent of oxidation on aromatic amino acids and are more susceptible to be indirectly oxidized by lipid peroxidation products. Different from traditional measurement of protein carbonyls and thiols, proteomics-based strategy better characterizes the targeted oxidation sites within proteins. The future trends using more robust and accurate targeted proteomics, such as parallel reaction monitoring strategy, to characterize protein oxidation in aquatic products are also given.

Structural characteristics of high-moisture extrudates with oil-in-water emulsions.

There is an increasing demand to produce high-quality plant-based meat analogs rich in tenderness and juiciness, presenting a significant challenge in creating oil-containing fibrous structures. A novel oil addition approach was developed by adding oil-in-water (O/W) emulsion during high-moisture extrusion processing. The current study investigated the effect of oil content using O/W emulsion on high-moisture extrudates prepared from soy protein isolate (SPI) and wheat gluten (WG) (SPI-WG). The oil content in fibrous SPI-WG extrudate could be up to 8.0% using O/W emulsion, whereas only 4.0% was possible by direct oil addition. O/W emulsion addition significantly decreased the extrusion response parameters of die pressure and specific mechanical energy. Confocal laser scanning microscopy indicated that oil was distributed to small droplets (0.5-15.0 µm) within the protein matrix in SPI-WG extrudates. Oil-free SPI-WG extrudate presented a close-meshed protein network. In contrast, higher oil contents led to more porous structures in SPI-WG extrudates with 3.0-8.0% oil. O/W emulsion addition reduced the rubber-like texture and rheological properties of SPI-WG extrudates, which improved the textural attributes (e.g., chewiness), making them similar to those of cooked chicken breast. In addition, the mobility of water and oil protons increased with increasing oil contents in SPI-WG extrudates, indicating the water and oil binding properties were reduced. Overall, this study demonstrated the potential of using O/W emulsions to promote fibrous structures of high-moisture extrudates.

Sodium chloride-induced oxidation of bighead carp (Aristichthys nobilis) fillets: The role of mitochondria and underlying mechanisms.

The NaCl addition is associated closely with oxidation of meat, and mitochondria are the key regulator in oxidation process of proteins. This study was set out to find a potential mechanism of oxidation induced by different concentrations NaCl (0 M, 0.5 M, 1.0 M and 2.0 M) based on the mitochondria pathway in fish. With the increase in concentrations of NaCl, the oxidation levels in bighead carp fillets moved to a higher degree. Samples showed significant lower mitochondrial membrane potential and permeability than control after being treated with 1.0 M and 2.0 M NaCl for 3 h. The activities of succinate dehydrogenase and cytochrome-c reductase decreased significantly in 0.5 M, 1.0 M and 2.0 M NaCl-treated samples and reactive oxygen species content increased in 2.0 M NaCl-treated samples compared to 0 M group. NaCl also destroyed the structure of the mitochondrial membranes and together influenced the activities of peroxisomes. Overall, 2.0 M NaCl promotes the lipid or protein oxidation in fillets might due to its effects on structure, complexes and antioxidant enzymes activity in mitochondria.

Exploration of the roles of spoilage bacteria in degrading grass carp proteins during chilled storage: A combined metagenomic and metabolomic approach.

Protein degradation1 caused by spoilage bacteria is highly related to fish quality deterioration during chilled storage. However, the exact roles of bacteria in degrading grass carp proteins are not fully known. In this work, we used metagenomics to analyze the microbiota composition in grass carp fillets, construct protein degrading pathways, and calculate taxonomic contributions to protein degrading functions. Besides, three dominant bacteria species were isolated and inoculated into sterile grass carp flesh, respectively. LC-MS-based untargeted metabolomics was then used to detect protein degradation metabolites produced by the inoculated bacteria. Combining the results of metagenomics and metabolomics, we found 1) Shewanella putrefaciens was active in hydrolyzing fish proteins (especially collagens) and produced quantities of putrescine through the arginine decarboxylase pathway; 2) Pseudomonas putida had potent potentials in utilizing oligopeptides and free amino acids; 3) Serine was a potential energy source for microbial growth and it was largely consumed by spoilage bacteria; 4) S. putrefaciens could form a metabiosis relationship with P. putida, due to their complementary roles in degrading fish proteins. Finally, we concluded that S. putrefaciens had the strongest spoilage potential among tested bacteria, suggesting the importance of S. putrefaciens inhibition in fish quality preservation. Meanwhile, this study contributed to a better understanding of microbial roles in fish spoilage and provided useful information for the development of new preservation methods.

Proteomic analysis of exudates in thawed fillets of bighead carp (Hypophthalmichthys nobilis) to understand their role in oxidation of myofibrillar proteins.

For frozen fillets, the formation of ice crystals destroys the integrity of cell and organelle membranes and causes the release of enzymes that are capable of catalyzing oxidation of myofibrillar proteins (MPs). Exudates from fresh, freeze-thaw (F-T) treated, and frozen stored fillets that were contained those enzymes were collected to explore the protein composition and changes in abundance of the main protein oxidation-related enzymes. Results indicated that enzymes with oxidative capacity were up-regulated and some antioxidant enzymes were down-regulated in exudates collected from 5 months frozen fillets. Changes in abundance of MPs in exudates suggested that degradation of MPs in thawed fillets was a comprehensive result of the F-T treatment, enzymatic degradation, and protein oxidation. The oxidative capacity of exudates was confirmed because incubation with exudates enhanced carbonyls and Schiff bases contents in MPs. Overall, the results of our study suggested that enzymes in exudates were a potential factor in protein oxidation in thawed fillets.

Current progress in kokumi-active peptides, evaluation and preparation methods: a review.

Kokumi is a complex sensation characterized by thickness, mouthfulness and continuity. Kokumi-active peptides, which are distributed in many kinds of food, induce a rich and long-lasting mouthfeel of food. Aimed to provide a comprehensive overview of kokumi peptides, this review covers the aspects of preparation and evaluation methods for kokumi peptides, kokumi receptor calcium-sensing receptor (CaSR), as well as structural features of kokumi peptides and derivatives. Apart from extraction and separation from natural and fermented food, preparation of kokumi peptides can be effectively obtained from enzymatic generation. Kokumi peptides are perceived by CaSR in taste cells and the proposed transduction pathway has been described. The evaluation on kokumi-inducing effect of peptides has employed a combination of sensory assessment and CaSR method. The discovered kokumi peptides mainly comprise glutamyl peptides, leucyl peptides and other peptides without specific features. Derivatives of amino acids and peptides including sulphur-containing amino acids, N-acyl-Tyr derivatives, N-acetylated amino acids and Maillard reaction products (MRPs) also work as kokumi enhancers. Based on the summarized developments, great sensory properties and bioactivities enable kokumi peptides as promising protein ingredients in future application.

Lignans from Euphorbia hirta L.

Five new lignans, euphorhirtins A-D (1-4), 5-methoxyvirgatusin (5), three artefacts, 7S-ethoxyisolintetralin (6), 7R-ethoxyisolintetralin (7), and 7R-ethoxy-3-methoxyisolintetralin (8), together with 13 known ones (9-21) were isolated from the medicinal plant Euphorbia hirta L. The structures of the compounds were elucidated by means of extensive spectroscopic analysis, including 1D and 2D NMR and HR-ESI-MS experiments. The absolute configurations of compound 1 was determined by ECD calculation. The isolates were evaluated for their inhibitory effects against the proliferation of the cancer cell lines (Hep G2, A549, and DU145) and compounds 14 and 18 showed inhibitory activity against the Hep G2 cells with IC50 values 7.2 ± 0.17 and 8.5 ± 0.36 µM.

Effect of protein oxidation in meat and exudates on the water holding capacity in bighead carp (Hypophthalmichthys nobilis) subjected to frozen storage.

This study aimed to investigate the effect of myofiber changes and protein oxidation on water holding capacity (WHC) of bighead carp fillets stored at -20 °C. WHC, microstructure, protein oxidation parameters, and specific modifications of oxidized amino acids were analyzed during 9 months of frozen storage. Results indicated that WHC decreased accompanied by myofibers' structural changes (including the formation of cavities among myofibers, breakage of myofibrils and myofibers, and shortening of sarcomeres) and protein oxidation. SDS-PAGE and carbonyl and sulfhydryl content determination of myofibrillar proteins and exudates gave a detailed description of the protein oxidation. LC-MS/MS analysis demonstrated that oxidation, di-oxidation, and 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) adduction were the top four oxidative modifications of proteins. Oxidative modifications impaired configuration and polarity of proteins, which may further affect WHC. This study provides plausible explanations to support the role of protein oxidation in the decrease of WHC in frozen fillets.

Modeling relationship between protein oxidation and denaturation and texture, moisture loss of bighead carp (Aristichthys Nobilis) during frozen storage.

To evaluate the effects of protein oxidation and denaturation on the fish texture and moisture loss during frozen storage, we measured the changes of protein oxidation and denaturation (salt-soluble protein (SSP), total sulfhydryl (SH), disulfide (SS), carbonyl contents and Ca2+ -ATPase activity), texture (hardness), and moisture loss (drip loss) of bighead carp fillets stored at -12, -20 and -28°C during 16 weeks. These data were employed to develop partial least squares regression (PLSR) model, radial basis function neural network (RBFNN) model, PLSR-RBFNN (PR) model and RBFNN-PLSR (RP) model. The results showed that the RP model provided no enhancement to RBFNN model because it had the exactly same root mean square error (RMSE) and R2 . PLSR model showed better performance than other models when predicting hardness. More appropriate linear or linearity-dominant hybrid model needed to be explored to establish the relationship between protein oxidation and denaturation and texture. The PR model performed better than other models in predicting drip loss with its lower RMSE and higher R2 , which revealed both linear and nonlinear relationship between protein oxidation and denaturation and moisture loss. Therefore, the PR model was a promising and encouraging tool to provide a more comprehensive understanding of the relationship between protein oxidation and denaturation and moisture loss of fish during frozen storage. PRACTICAL APPLICATION: The study explored the effects of protein oxidation and denaturation on the texture and moisture loss of bighead carp during frozen storage (-12 to -28°C). PLSR model showed better performance than other models when predicting the relationship between protein oxidation and denaturation and texture. The PR model was an available tool for manufacturers to predict the relationship between protein oxidation and denaturation and moisture loss.

Tracking structural modifications and oxidative status of myofibrillar proteins from silver carp (Hypophthalmichthys molitrix) fillets treated by different stunning methods and in vitro oxidizing conditions.

In order to explain the increased susceptibility of stunning-stressed fillets to oxidative modifications, effect of stunning methods (percussion and gill cut) and in vitro metal-catalyzed oxidation on structural changes and oxidative status of myofibrillar proteins (MPs) from silver carp fillets was examined. In comparison to the percussion group, oxidized MPs (10 mM H2O2) from gill cut-stunned fillets exhibited higher extent of structural disintegration as well as elevated carbonyl levels. Using label-free proteomics, isoforms of myosin heavy chain and actin were major proteins underwent oxidative modifications including monooxidation of methionine, dioxidation of aromatic amino acids, adduction of lipid peroxidation products with aliphatic amino acids, and the carbonylation of lysine and arginine into semialdehydes. In addition, amino acids located at the tail portion of myosin were highly accessible to oxidation. Owing to the structural disorganization caused by stunning stress, MPs from gill cut-stunned fillets were more susceptible to oxidation in vitro.

Purification and characterization of antioxidant peptides from yak (Bos grunniens) bone hydrolysates and evaluation of cellular antioxidant activity.

In this study, papain and alcalase were used to generate antioxidant peptides from yak bone protein. The antioxidant activities of hydrolysates in vitro were evaluated by 2,2'-azinobios-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity, total reducing power, ferrous ion chelating ability and hydroxyl radical scavenging activity. The hydrolysates generated by alcalase possessed the best antioxidant activity among unhydrolyzed protein and samples treated by papain, but the antioxidant activity decreased after simulated gastrointestinal digestion in vitro. The products of simulated gastrointestinal digestion were separated by ultrafiltration and high performance liquid chromatography, and the amino acid sequences of peptides were identified by mass spectrometry. The digestion sites within peptides were predicted by a bioinformatics strategy, and ten peptides were selected for synthesis. Among 10 synthetic peptides, Gly-Phe-Hyp-Gly-Ala-Asp-Gly-Val-Ala, Gly-Gly-Pro-Gln-Gly-Pro-Arg and Gly-Ser-Gln-Gly-Ser-Gln-Gly-Pro-Ala possessed strong antioxidant activities, among which Gly-Phe-Hyp-Gly-Ala-Asp-Gly-Val-Ala had a significant cytoprotective effect in Caco-2 cells under oxidative stress induced by H2O2, which reduced the formation of reactive oxygen species and malondialdehyde, and improved the activity of antioxidant enzymes in cells. These results showed that yak bone peptides exhibited strong antioxidant activity and have a potential value as a new type of natural antioxidant.

Spoilage-related microbiota in fish and crustaceans during storage: Research progress and future trends.

Fish and crustaceans are highly perishable due to microbial growth and metabolism. Recent studies found that the spoilage process of fish and crustaceans is highly related to their microbiota composition. Microbiota of fish and crustaceans changes dramatically during storage and can be influenced by many factors (e.g., aquaculture environment, handling process, storage temperature, and various quality control techniques). Among them, many quality control techniques have exhibited efficient effects on inhibiting spoilage bacteria, regulating microbiota composition, and retarding quality deterioration. In this article, we elucidate the relationship between microbiota composition and fish/crustacean spoilage, demonstrate influencing factors of fish/crustaceans microbiota, and review various quality control techniques (especially plant-derived preservatives) including their preservative effects on microbiota and quality of fish and crustaceans. Besides, present and future trends of various detective methods used in microbiota analysis are also compared in this review, so as to provide guides for future microbiota studies. To conclude, novel preservation techniques (especially plant-derived preservatives) and hurdle technologies are expected to achieve comprehensive inhibitory effects on spoilage bacteria. Efficient delivery systems are promising in improving the compatibility of plant-derived preservatives with fish/crustaceans and enhancing their preservative effects. Besides, spoilage mechanisms of fishery products that involve complex metabolisms and microbial interactions need to be further elucidated, by using omics technologies like metagenomics, metatranscriptomics, and metabolomics.