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Sixteen female pigs (Duroc x Landrace x Large White) were divided into 2 groups, which had either free access to the diet (control group) or were feed-restricted from d 28 to 80 and then had free access to the diet (compensatory growth group). The sensory analysis showed that the pigs exhibiting compensatory growth produced meat with increased tenderness compared with control pigs (P < 0.05). To gain further knowledge of the influence of compensatory growth on meat tenderness, the sarcoplasmic protein fraction of muscle tissue was studied at the time of slaughter and 48 h postmortem using proteome analysis. At slaughter, 7 different proteins were found to be affected by compensatory growth: HSC70, HSP27, enolase 3, glycerol-3-phosphate dehydrogenase, aldehyde dehydrogenase E2, aldehyde dehydrogenase E3, and biphosphoglycerate mutase. The HSC70 and HSP27 both belong to the heat shock family and are known to play a role during muscle development. Hence, they may be affected by compensatory growth and increased protein turnover. Forty-eight hours after slaughter, 8 different proteins were found to be affected by compensatory growth: myosin light chain (MLC) II, MLC III, sulfite oxidase, chloride intracellular channel 1, 14-3-3 protein % elongin B, and phosphohistidine phosphatase 14. The changes observed on MLC II and MLC III could be a consequence of enzymatic cleavage in the neck region of the globular myosin head domain that causes the release of MLC II and MLC III from the actomyosin complex. It has previously been hypothesized that compensatory growth results in an increased postmortem proteolysis; thus it was presumed that the intensity of some protein fragments would be affected by compensatory growth. However, the peptides that were found to be affected at 48 h postmortem were all full-length proteins. The 14-3-3 protein [gamma] has been proposed to play a role in the contraction of muscle during rigor and may thereby have an effect on meat tenderness. This study reveals some very interesting changes in the muscle proteome affected by compensatory growth, which may be useful in understanding the relationship among compensatory growth, protein turnover, and meat tenderness. Key words: compensatory growth, feeding strategy, meat quality, muscle growth, proteome analysis, sarcoplasmic protein

Abstract Background During the transmission route from poultry to the human host, the major foodborne pathogen C. jejuni may experience many types of stresses, including low pH caused by different acids. However, not all strains are equally sensitive to the stresses. The aim of this study was to investigate the response to acid stress of three sequenced C. jejuni strains with different acid tolerances using HCl and acetic acid. Results Two-dimensional gel electrophoresis was used for proteomic analysis and proteins were radioactively labelled with methionine to identify proteins only related to acid exposure. To allow added radioactive methionine to be incorporated into induced proteins, a modified chemically defined broth was developed with the minimal amount of methionine necessary for satisfactory growth of all strains. Protein spots were analyzed using image software and identification was done with MALDI-TOF-TOF. The most acid-sensitive isolate was C. jejuni 327, followed by NCTC 11168 and isolate 305 as the most tolerant. Overall, induction of five proteins was observed within the pI range investigated: 19 kDa periplasmic protein (p19), thioredoxin-disulfide (TrxB), a hypothetical protein Cj0706 (Cj0706), molybdenum cofactor biosynthesis protein (MogA), and bacterioferritin (Dps). Strain and acid type dependent differences in the level of response were observed. For strain NCTC 11168, the induced proteins and the regulator fur were analysed at the transcriptomic level using qRT-PCR. In this transcriptomic analysis, only up-regulation of trxB and p19 was observed. Conclusions A defined medium that supports the growth of a range of Campylobacter strains and suitable for proteomic analysis was developed. Mainly proteins normally involved in iron control and oxidative stress defence were induced during acid stress of C. jejuni. Both strain and acid type affected sensitivity and response.

Abstract Background Aspergillus niger is a filamentous fungus found in the environment, on foods and feeds and is used as host for production of organic acids, enzymes and proteins. The mycotoxin fumonisin B2 was recently found to be produced by A. niger and hence very little is known about production and regulation of this metabolite. Proteome analysis was used with the purpose to reveal how fumonisin B2 production by A. niger is influenced by starch and lactate in the medium. Results Fumonisin B2 production by A. niger was significantly increased when lactate and starch were combined in the medium. Production of a few other A. niger secondary metabolites was affected similarly by lactate and starch (fumonisin B4, orlandin, desmethylkotanin and pyranonigrin A), while production of others was not (ochratoxin A, ochratoxin alpha, malformin A, malformin C, kotanin, aurasperone B and tensidol B). The proteome of A. niger was clearly different during growth on media containing 3% starch, 3% starch + 3% lactate or 3% lactate. The identity of 59 spots was obtained, mainly those showing higher or lower expression levels on medium with starch and lactate. Many of them were enzymes in primary metabolism and other processes that affect the intracellular level of acetyl-CoA or NADPH. This included enzymes in the pentose phosphate pathway, pyruvate metabolism, the tricarboxylic acid cycle, ammonium assimilation, fatty acid biosynthesis and oxidative stress protection. Conclusions Lactate added in a medium containing nitrate and starch can increase fumonisin B2 production by A. niger as well as production of some other secondary metabolites. Changes in the balance of intracellular metabolites towards a higher level of carbon passing through acetyl-CoA and a high capacity to regenerate NADPH during growth on medium with starch and lactate were found to be the likely cause of this effect. The results lead to the hypothesis that fumonisin production by A. niger is regulated by acetyl-CoA.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study aimed to determine the compatibility of pre-fermented sugar beet pulp to support the growth of Pleurotus ostreatus mycelium in submerged fermentation. The goal was to create a meat alternative based on mycelial-fermented pulp. It was further explored whether pre-fermentation with lactic acid bacteria (LAB) on the pulp increased meat-like properties, such as aroma, springiness, and hardness, in the final product. Three strains were selected from a high throughput screening of 105 plant-derived LAB based on their acidification and metabolite production in the pulp. Two homofermentative strains (Lactococcus lactis) and one heterofermentative strain (Levilactobacillus brevis) were selected based on their low ethanol production, high lactic acid production, and overall acidification of the pulp. Mycelium of P. ostreatus was grown in submerged fermentations on the pre-fermented pulp, and the biomass was removed by centrifugation. The fungal strain consumed all available sugars and acids and released arabinose to the media. Volatiles were detected using GC-MS, and a large increase in concentrations of hexanal, 1-octen-3-ol, and 2-octenal was measured. Concentration of 1-octen-3-ol was lower in the pre-fermented samples vs. the non-pre-fermented. LC-MS amino acid analysis showed the presence of all essential amino acids on day 0 and 7 of fermentation. The highest concentration of amino acids was for glutamic acid/glutamine and aspartic acid/asparagine. A decrease in all amino acids after 7 days of fungal fermentation was measured for all fermentations. The decrease was more significant for pre-fermented samples. This was also confirmed through a total protein determination, except for samples pre-fermented with Lactococcus lactis strain NFICC142 which increased in total protein content after fungal fermentation. The protein digestibility increased after fungal fermentation, and the highest increase was seen for non-pre-fermented samples. The springiness of the fermented product indicated similarities to meat alternatives, while the hardness was much lower than other meat alternatives. The results indicate that dried sugar beet pulp can be used for submerged cultivation of P. ostreatus, but that pre-fermentation does not improve the physical or nutritional properties of the end product significantly, except for an increased protein content for NFICC142 pre-fermented media. This is the first known attempt to use LAB and P. ostreatus in mixed fermentation to produce fungal mycelium, as well as the first attempt at using SBP in a liquid fermentation for mycelial production of P. ostreatus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

This study aimed to illustrate the relationship among physicochemical properties, subunit composition and protein functionalities in a broad collection of commercial soy protein isolates (SPIs) from China and the EU. The results indicated that SPIs had large variations in glycinin/β-conglycinin composition, protein denaturation, and water- and oil-binding capacity (WBC and OBC) and solubility. These SPIs could be roughly divided into pre-denatured SPI, partially hydrolyzed SPI, and less modified SPI. The pre-denatured SPI with high surface hydrophobicity and large particle sizes showed reduced WBC and OBC due to increased protein aggregation, and partially hydrolyzed SPI showed high protein solubility owing to the exposure of hydrophilic regions and reduction in molecular size. The processing-induced physicochemical changes played a pivotal role in determining protein functionalities, whereas subunit composition affected protein functionality less. Overall, this study highlighted the obvious difference in raw material quality of commercial SPI, and provided promising methods for SPI categorization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protein degradation and oxidation are two major alterations during the storage of processed bighead carp fillets. This study conducted a comparative analysis of degraded and oxidized products as well as oxidation sites in fresh, frozen and brined frozen bighead carp fillets. Frozen storage played a dominant role in protein degradation and oxidation, and brining promoted these changes. In brined frozen samples, the decreased SDS-PAGE band intensities for tropomyosin, troponin, and myosin light chain were mainly due to their degradation. Myosin heavy chain fast skeletal muscle was the most oxidized and degraded protein during storage, with modifications such as monooxidation, protein-lipid peroxidation adducts, and α-aminoadipic semialdehydes formation. Amino acids in the tail portion of myosin were prone to oxidation than the head portions. Our results provided comprehensive insights into protein degradation and oxidation in bighead carp during storage, helping to assess the specific fate of oxidative products in future dietary investigations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

The typical dry-cured ham flavor is rich in umami and brothy perceptions, for which short peptides may contribute. Particularly, γ-glutamyl peptides could be the responsible of these previously reported attributes, as they exert a synergistic interaction with other basic tastes and modify the intensity of salty, sweet, and umami tastes. The content of peptides has been reported to evolve along the processing, but no kokumi γ-glutamyl peptides have been identified in Spanish dry-cured hams yet. In this research, nine γ-glutamyl dipeptides (γ-EA, γ-EC, γ-EE, γ-EF, γ-EL, γ-EM, γ-EV, γ-EW, and γ-EY) and two γ-glutamyl tripeptides (GSH and γ-EVG) have been quantitated at 6, 12, 18 and 24 months of traditional processing of Spanish dry-cured ham by performing a Q Exactive Orbitrap-based tandem mass spectrometry. The results show an increase of γ-EA, γ-EE, γ-EF, γ-EL, γ-EM and γ-EVG, obtaining maximums at 24 months of curing ranging from 0.14 (γ-EVG) to 18.86 (γ-EL) μg/g dry-cured ham. Otherwise, γ-EV, γ-EW and γ-EY accumulated until the 18th month of storage to 15.10, 0.54 and 3.17 μg/g dry-cured ham, respectively; whereas γ-EC and GSH amounts decreased starting from 0.0676 and 4.41 μg/g dry-cured ham, respectively at earlier stages. The concentration dynamics of these compounds may be linked with proteolytic and oxidative reactions during processing. In addition, due to their synergistic effect on kokumi activity, this could constitute insights of the brothy perceptions of dry-cured ham, and these peptides probably contribute to the sensory differences existing in long processed Spanish dry-cured hams., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2023 The Authors. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

Salting is a crucial step during the production of dry-cured ham and it is not well known whether it has an impact on the generation of taste-active peptides. The present study focused on the quantitation of kokumi γ-glutamyl peptides in low-salted Spanish dry-cured hams with 12 months of processing. By using mass spectrometry, peptides were quantitated from samples obtained after ethanolic deproteinization-based and non-ethanolic deproteinization-based extraction methods. Peptides γ-EA, γ-EE, and γ-EL registered mean values of 0.31, 2.75, and 11.35 µg/g of dry-cured ham, respectively, with no differences observed between both extraction protocols. However, γ-EF, γ-EM, γ-EV, γ-EW, γ-EY, and γ-EVG presented significantly ( p < 0.05) higher concentrations in the ethanolic deproteinized samples showing values of 5.58, 4.13, 13.90, 0.77, 3.71, and 0.11 µg/g of dry-cured ham, respectively. These outcomes reflect the importance of protocols for the extraction of peptides to achieve the most feasible results. In addition, potential precursors for the formation of γ-glutamyl peptides are generated during dry-curing under salt restriction. The kokumi activity of these γ-glutamyl peptides could enhance the sensory attributes countering the taste deficiencies caused by the salt restriction.

As the plant-based food market grows, demand for plant protein is also increasing. Proteins are a major component in foods and are key to developing desired structures and textures. Seed storage proteins are the main plant proteins in the human diet. They are abundant in, for example, legumes or defatted oilseeds, which makes them an excellent candidate to use in the development of novel plant-based foods. However, they often have low and inflexible functionalities, as in nature they are designed to remain densely packed and inert within cell walls until they are needed during germination. Enzymes are often used by the food industry, for example, in the production of cheese or beer, to modify ingredient properties. Although they currently have limited applications in plant proteins, interest in the area is exponentially increasing. The present review first considers the current state and potential of enzyme utilization related to plant proteins, including uses in protein extraction and post-extraction modifications. Then, relevant opportunities and challenges are critically discussed. The main challenges relate to the knowledge gap, the high cost of enzymes, and the complexity of plant proteins as substrates. The overall aim of this review is to increase awareness, highlight challenges, and explore ways to address them.

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl- p -nitroanilide (γ-G p NA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-G p NA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.

This study aimed to characterize the substrate affinity and catalytic efficiency of bovine milk γ-glutamyltransferase (BoGGT) towards different donors and acceptors by comparing it with a reference (Bacillus amyloliquefaciens, BaGGT). Quantitation of γ-glutamyl peptides and free amino acids was conducted in combination with enzymatic kinetic. Kokumi peptides were generated from whey protein hydrolysates through transpeptidation catalyzed by both GGTs. BaGGT has a higher transpeptidase activity than BoGGT when γ-glutamyl-p-nitroanilide (γ-GpNA) or glutamine acts as a donor. Glutamine is a better γ-glutamyl donor than γ-GpNA for both GGTs. Furthermore, membrane-free BoGGT has a more efficient activity and higher substrate affinity than the native BoGGT. BoGGT has the highest affinity with Val-Gly and can produce γ-Glu-Val-Gly, a substance with a strong kokumi intensity and the lowest taste threshold. This study reveals that the catalytic ability of GGT is highly dependent on the acceptor, and membrane interactions restrict the transpeptidase activity of BoGGT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)