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.

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.

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.

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.

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.

Dolabellane and Clerodane Diterpenoids from the Twigs and Leaves of Casearia kurzii.

A pair of enantiomeric 15-nordolabellane diterpenoids, (-)- and (+)-caseadolabellols A (1a and 1b), three dolabellane diterpenoids, caseadolabellols B-D (2-4), two dolastane diterpenoids, caseadolastols A and B (5 and 6), 10 clerodane diterpenoids, caseakurzins A-J (7-16), and nine known diterpenoids (17-25) were isolated from the twigs and leaves of Casearia kurzii. The structures of the new compounds were established on the basis of extensive spectroscopic data, and those of compounds 1a, 1b, and 2 were verified by single-crystal X-ray crystallographic analysis. The enantiomers 1a and 1b were separated by chiral-phase HPLC. The absolute configurations were determined by experimental and calculated ECD data, the modified Mosher's method, or literature comparison. Compounds 1a and 5 showed significant quinone reductase-inducing activity in Hepa 1c1c7 cells, while 1b showed moderate activity. Molecular docking studies showed that 1a had greater binding affinity with Nrf2 protein (5FNQ) than 1b. The cytotoxic activity of compounds 1a, 1b, 2-12, 15, and 16 was evaluated, among which compounds 8 and 16 exhibited significant inhibitory activity against the A549 cell line. Compounds 8 and 16 induced the A549 cells to arrest at G2/M and S phases, respectively, and both compounds induced apoptosis in A549 cells.

Assessment of bacterial contributions to the biochemical changes of chill-stored blunt snout bream (Megalobrama amblycephala) fillets: Protein degradation and volatile organic compounds accumulation.

In this study, we evaluated the contributions of three bacteria (Pseudomonas versuta, Shewanella putrefaciens, and Aeromonas sobria) to the proteolysis, biogenic amines formation, volatile organic compounds accumulation, lipid oxidation, nucleotide catabolism, discoloration, and water migration of bream flesh during chilled storage. The results showed that P. versuta exhibited hydrolyzing activity against sarcoplasmic proteins, and all three strains could degrade myofibrillar proteins, specifically actin. The highest producer of putrescine was S. putrefaciens, which reached a maximum level 5.05 mg/kg after 14 days. Compared with the A. sobria group, hypoxanthine riboside degraded faster in samples inoculated with P. versuta or S. putrefaciens, A. sobria, P. versuta, and S. putrefaciens were responsible for the production of alcohol and aldehydes, whereas only S. putrefaciens produced thiophene and partial esters. Fish flesh inoculated with P. versuta, S. putrefaciens, and A. sobria presented slight green, yellow, and pink discoloration, respectively.

Effect of grape seed extract on quality and microbiota community of container-cultured snakehead (Channa argus) fillets during chilled storage.

Herein, the effects of grape seed extract (GSE) on the microflora and biochemical changes of container cultured snakehead (Channa argus) fillets during 11 days of chilled storage were investigated. The sensory analysis, the total number of viable colonies, the total amount of volatile basic nitrogen, and k-value analysis revealed that GSE retarded the deterioration of snakehead fillets. The degradation of inosine 5'-monophosphate and the accumulation of inosine and hypoxanthine in the GSE group were slower than these in the control group. Moreover, GSE treatment effectively decreased the accumulation of putrescine, cadaverine, and histamine. Illumina-MiSeq high throughput sequencing results showed that GSE inhibited the growth of Aeromonas on snakehead fillets. Based on the microbial enumeration, sensory analysis, and k-value, GSE prolonged the shelf life of fillets for 3 days, suggesting its potential for snakehead fillets preservation.

Biochemical changes induced by dominant bacteria in chill-stored silver carp (Hypophthalmichthys molitrix) and GC-IMS identification of volatile organic compounds.

To evaluate the spoilage potential of dominant bacteria (Aeromonas allosaccharophila, Pseudomonas psychrophila, and Shewanella putrefaciens) isolated from spoiled silver carp (Hypophthalmichthys molitrix) fillets, biochemical changes including protein degradation, trichloroacetic acid (TCA)-soluble peptides, total volatile basic nitrogen (TVB-N), biogenic amines, nucleotide catabolism, and volatile organic compounds were examined in single-species inoculated silver carp flesh for 14 days at 4 °C. P. psychrophila exhibited the strongest proteolytic activity, which resulted in the highest concentrations of TCA-soluble peptides and TVB-N. S. putrefaciens was responsible for the production of putrescine and cadaverine and led to the fastest degradation of hypoxanthine riboside (HxR). At the end of storage, P. psychrophila was the main producer of ketones, especially the C7-C9 ketones, while sulfur compounds were released primarily by S. putrefaciens. Moreover, 1-propanol, butanone, 2-hexanone, methyl isobutyl ketone, dimethyl sulfide, and dimethyl disulfide increased gradually with storage time, suggesting their potential as spoilage markers for freshness/spoilage monitoring. P. psychrophila possessed the strongest spoilage potential in the fish matrix, followed by S. putrefaciens, whereas A. allosaccharophila showed a very low spoilage potential. In conclusion, P. psychrophila and S. putrefaciens were identified as the specific spoilage organisms (SSOs) of silver carp, suggesting that preservation researchers should focus on these two spoilage contributors in future studies. This research contributes to a deeper understanding of silver carp spoilage and to the development of methods and tools to improve fish quality management.

Changes in microbial communities and quality attributes of white muscle and dark muscle from common carp (Cyprinus carpio) during chilled and freeze-chilled storage.

This study investigated sensory scores, quality attributes and microbial communities in white muscle and dark muscle of common carp (Cyprinus carpio) during chilled (4 °C) and freeze-chilled (-20 °C for 4 weeks prior to 4 °C) storage. Compared to the samples at the end of storage, fresh samples and frozen-thawed samples on day 0 showed greater bacterial diversity and more differences in microbiota. Initially, Aeromonas was the prevalent genus in fresh white muscle and dark muscle. As time progressed, Aeromonas followed by Pseudomonas predominated in white muscle, while Aeromonas, Pseudomonas, and Lactococcus dominated in dark muscle. Paenibacillus was identified as the largest population in frozen-thawed samples of both muscle types, but Pseudomonas increased dramatically to become dominant in the two spoiled samples. Volatile organic compounds (VOCs) of carp muscle consisted mainly of aldehydes and alcohols, and the percentage of ketones in both muscle types increased considerably after storage. Moreover, dark muscle showed more kinds of VOCs, and a slower rate of quality deterioration than white muscle. Based on sensory assessment, the shelf-life of white muscle and dark muscle of common carp for chilled storage was 8 days and 10 days, respectively, as well as 8 days together for freeze-chilled storage.

Application of Illumina-MiSeq high throughput sequencing and culture-dependent techniques for the identification of microbiota of silver carp (Hypophthalmichthys molitrix) treated by tea polyphenols.

This study evaluated the antimicrobial effects of tea polyphenols (TP) on changes in microbiota composition and quality attributes in silver carp fillets stored at 4 °C. During storage, TP treatment was found to be effective in enhancing sensory quality, inhibiting microbial growth, and attenuating chemical quality deterioration. Meanwhile, the composition of microbiota of silver carp fillets was investigated using culture-dependent and culture-independent methods. Initially, compared to the control, TP obviously decreased the relative abundance of Aeromonas, which allowed Acinetobacter and Methylobacterium to become the dominant microbiota in TP treated fillets on day 0. The controls, 0.5% TP-treated fillets, and 1% TP-treated fillets were rejected by sensory panelists on days 8, 12, and 12, respectively. At the time of sensory rejection, Aeromonas, followed by Acinetobacter and Pseudomonas, became the main spoilers in the control on day 8. However, TP treatment inhibited the growth of Aeromonas and Acinetobacter significantly. Consequently, Aeromonas followed by Pseudomonas and Shewanella became the predominant microbiota in all TP-treated fillets on day 12. Therefore, TP improved the quality of fillets during chilled storage, which was mainly due to their modulating effects on microbiota that resulted in the change in pattern and process of spoilage in fillets.

Botrysphones A-C and Botrysphins A-F, Triketides and Diterpenoids from the Fungus Botrysphaeria laricina.

Three new triketides, botrysphones A-C (1-3) and six new isopimarane-type diterpenoids, botrysphins A-F (4-9), together with the known triketides sphaeropsidone (10) and chlorosphaeropsidone (11) and diterpenoids sphaeropsidins A and B (12 and 13), were obtained from culture of the fungus Botrysphaeria laricina associated with the moss Rhodobryum umgiganteum. The structures of the new compounds were established on the basis of extensive spectroscopic techniques including HRMS and 1D and 2D NMR data. Compounds 7 and 12 showed significant quinone reductase inducing activity in Hepa 1c1c7 cells.

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.

Effect of Thermal Treatment on Gelling and Emulsifying Properties of Soy ß-Conglycinin and Glycinin.

This study investigated the impact of different preheat treatments on the emulsifying and gel textural properties of soy protein with varying 11S/7S ratios. A mixture of 7S and 11S globulins, obtained from defatted soybean meal, was prepared at different ratios. The mixed proteins were subjected to preheating (75 °C, 85 °C, and 95 °C for 5 min) or non-preheating, followed by spray drying or non-spray drying. The solubility of protein mixtures rich in the 7S fraction tended to decrease significantly after heating at 85 °C, while protein mixtures rich in the 11S fraction showed a significant decrease after heating at 95 °C. Surprisingly, the emulsion stability index (ESI) of protein mixtures rich in the 7S fraction significantly improved twofold during processing at 75 °C. This study revealed a negative correlation between the emulsifying ability of soy protein and the 11S/7S ratio. For protein mixtures rich in either the 7S or the 11S fractions, gelling proprieties as well as emulsion activity index (EAI) and ESI showed no significant changes after spray drying; however, surface hydrophobicity was significantly enhanced following heating at 85 °C post-spray drying treatment. These findings provide insights into the alterations in gelling and emulsifying properties during various heating processes, offering great potential for producing soy protein ingredients with enhanced emulsifying ability and gelling property. They also contribute to establishing a theoretical basis for the standardized production of soy protein isolate with specific functional characteristics.

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.

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.

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.

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.

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.

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.