An unfolding/aggregation kinetic instructed rational design towards improving graft degree of glycation for myofibrillar protein.

Glycation is an effective strategy for the application of myofibrillar protein (MP) in beverage formulas by improving water solubility. In conventional glycation, the efficiency was limited as MP-saccharides conjugates mostly produced at low temperature due to thermosensitivity. This study was aimed to explore unfolding/aggregation kinetics of MP, including aggregate behavior, structural characteristics, and micromorphology, which guided the selection of temperature for glycation. It was shown that 40 °C/47.5 °C were critical temperature for MP unfolding/aggregation, respectively. Accordingly, an innovative technology of glycation (cyclic continuous glycation, CCG) was established by combining such temperatures. The results confirmed that cyclic continuous heating (CCH) inhibited excessive exposure of sulfhydryl and hydrophobic groups impeding protein aggregation. Importantly, it was revealed that rational designed CCG promoted covalent binding of MP to glucose by regulating unfolding-aggregation balance, exhibiting higher glycation degree. Overall, CCG-modified MP is expected to motivate the application of meat proteins in food formulations.

Insight into the mechanisms of combining direct current magnetic field with phosphate in promoting emulsifying properties of myofibrillar protein.

This study investigated the combined effects of direct-current magnetic field (DC-MF, 9.5 mT) and tetrasodium-pyrophosphate (TSPP, 1-5 g/L) on emulsified gel properties of porcine myofibrillar protein (MP). Results showed that MP at DC-MF and 3 g/L TSPP had decreased spectrum intensity of UV and fluorescence compared to that without DC-MF, owing to the changes of MP tertiary structure caused by DC-MF, especially tryptophan and tyrosine. The emulsion treated with DC-MF behaved better emulsifying activity and stability than that without DC-MF under such condition. And emulsion had lower creaming index and better storage stability. Gels prepared by this MP emulsion had low porosity and stable structure, accompanying with smaller size and more uniform distribution of oil droplets. Microstructure images showed that gels were covered with microporous structure, which was conducive to the good WHC of the emulsified gels (97.12%). These results showed the feasibility of DC-MF and TSPP in improving MP emulsion/emulsified gel.

Elucidating the mechanisms of ultrasound treatment combined with κ-carrageenan addition enhancing the gelling properties of heat-induced myofibrillar protein gel.

This work investigated the effect of ultrasound (US) treatment synergized with κ-carrageenan (KC) on the gel properties, structural characteristics and microstructures of myofibrillar protein (MP) gel. The results demonstrated that simply adding KC enhanced the gel strength and water holding capacity (WHC) of MP gels. Moreover, the gel strength and WHC of MP gels were increased by 56.67 % and 76.19 % via 20 min US treatment synergized with KC, which was mainly attributed to the changes in sulfhydryl content, surface hydrophobicity, and fluorescence intensity of MP gels. Based on the results of molecular docking and secondary structure, it can be hypothesized that the synergistic effect resulted in the rearrangement of the proteins, which altered the interaction site between MP gels and KC, accompanied by stronger binding. Furthermore, the microstructural results indicated that moderate US treatment (20 min) facilitated the production of a more compact and denser MP gels matrix with uniformly sized and distributed pores. However, excessive US treatment (40 and 50 min) caused the MP gels to form looser and disordered gel structure, which reduced the gel strength and WHC. This study suggested that combining of US and KC was a potential tactic to enhance the gelling properties of heat-induced MP gels.

A novel EGCG-Histidine complex improves gelling and physicochemical properties of porcine myofibrillar proteins: Insight into underlying mechanisms.

Herein, an EGCG-Histidine complex is prepared, characterized, and further used to improve gel properties of myofibrillar proteins (MP). Results of FTIR, XRD, UV-Vis spectroscopy showed that histidine is covalently bound to EGCG by Michael addition or Schiff base reaction to form EGCG-Histidine complex, and antioxidant activity of EGCG-Histidine complex is significantly increased compared to EGCG or histidine alone (P < 0.05). The addition of EGCG-Histidine complex results in cooking loss of gel decreasing from 66.7 ± 0.23 % to 40.3 ± 2.02 %, and improves rheological properties of MP, and enhances gel strength from 0.10 ± 0.01 N to 0.22 ± 0.03 N, indicating positive effect of EGCG-Histidine complex on MP gel formation, above results is supported by results of SEM, CD spectroscopy, SDS-PAGE, and tryptophan fluorescence. These results indicated that EGCG-Histidine complex can be used as a functional ingredient to improve gel quality of meat products.

Tracking protein aggregation behaviour and emulsifying properties induced by structural alterations in common carp (Cyprinus carpio) myofibrillar protein during long-term frozen storage.

The effect of ultrasound-assisted immersion freezing (UIF), air freezing (AF), and immersion freezing (IF) on the protein structure, aggregation, and emulsifying properties of common carp (Cyprinus carpio) myofibrillar protein during frozen storage were evaluated in the present study. The result showed that, compared with AF and IF samples, UIF sample had higher reactive/total sulfhydryl, protein solubility, and lower protein turbidity (P < 0.05), indicating that UIF was beneficial to inhibit protein oxidation and aggregation induced by frozen storage. UIF inhibited the alteration of secondary structure and tertiary structure during frozen storage. Meanwhile, UIF sample had higher emulsifying activity index, and smaller emulsion droplet diameter than AF and IF samples (P < 0.05), suggesting that UIF was beneficial for maintaining the emulsifying properties of protein during storage. In general, UIF is a potential and effective method to suppress the decrease in protein emulsifying properties during long-term frozen storage.

Effect of ultrasound treatment on porcine myofibrillar protein binding furan flavor compounds at different salt concentrations.

The effects of ultrasound (500 W) on the interaction of porcine myofibrillar protein (MP) with furan flavor compounds at different salt concentrations (0.6 %, 1.2 % and 2.4 %) were investigated. With the increase of salt concentration, the particle size of MP decreased, and the surface hydrophobicity and active sulfhydryl content increased due to the unfolding and depolymerization of MP. At the same time, ultrasound promoted the exposure of hydrophobic binding sites and hydrogen bonding sites of MP in different salt concentration systems, thus improving the binding ability of MP with furan compounds by 2 % to 22 %, among which MP had the strongest binding capacity of 2-pentylfuran. In conclusion, ultrasound could effectively promote the unfolding of the secondary structure of MP, which was beneficial to the combination of MP and furan flavor compounds under different salt concentrations.

Effect of MDA-mediated oxidation on the protein structure and digestive properties of golden pomfret.

In this study, golden pomfret myofibrillar protein (MP) was used as the research object, and the oxidation system of malondialdehyde (MDA) as an inducer and the static digestion model in vitro was established for the analysis of the changes in protein structure and molecular morphology during oxidation and digestion. Subsequently, the effects of MDA-mediated oxidation on the structure and digestive properties of golden pomfret myofibrillar fibrillar protein were determined. The results showed that the hydrolysis degree and digestion rate of MP were inhibited with the increase in MDA concentration (0, 0.5, 1, 2, 5, 10 mmol/L), and the carbonyl group, surface hydrophobicity, irregular curling, and MDA content increased significantly (P < 0.05), whereas the total sulfhydryl groups, α-helices, free amino groups, hydrolysis degree, and MDA incorporation decreased significantly (P < 0.05), The molecular particle size was significantly reduced (P < 0.05), and the molecular morphology and molecular structure were analyzed (P >0.05). Finally, the molecular size and cross-linking degree gradually increased. In conclusion, MDA can alter the structure and morphology of proteins, resulting in a decrease in hydrolysis and digestion rate. This study can provide theoretical support and reference for the regulation of protein digestion.

Properties of co-gel between Tenebrio Molitor larvae protein and myofibrillar protein induced by transglutaminase.

This study explored the effect of adding transglutaminase (TGase) to a co-gel of Tenebrio Molitor larvae protein (TMLP) and myofibrillar protein (MP). Different concentrations of TGase (0-90 U/g) were added to the co-gel. The results showed that 60 U/g TGase treatment significantly improved the gel strength and water holding capacity (WHC) by 26.51 g and 9.2 %, respectively. TGase promoted the rheological properties and accelerated the three-dimensional network structure of the co-gel. Moreover, TGase significantly increased (P < 0.05) the tyrosine residues, tryptophan residues content and hydrophobic interactions of the aliphatic groups. The chemical forces between the protein molecules changed. TGase promoted the transition of α-helix to ß-sheet and free water to immobilized water, thereby improving the WHC of co-gel. The principal component analysis reflected the links among indicators. This study illustrated that TGase might be an effective strategy to improve the co-gel of TMLP and MP and emulsified meat products with insects.

Oxidation affects pH buffering capacity of myofibrillar proteins via modification of histidine residue and structure of myofibrillar proteins.

The pH buffering capacity is an important functionality of muscle proteins, and muscle foods are susceptible to being oxidized during storage and processing. In order to study the effect of oxidation on the pH buffering capacity of myofibrillar proteins, myofibrils extracted from snakehead fish (Channa argus) were oxidized with H2O2. Results showed that increased oxidation led to loss of free sulfhydryl groups, formation of carbonyl groups, increased surface hydrophobicity, and aggregation of myofibrillar proteins. In addition, there was a significant reduction in the content of histidine in oxidized myofibrillar proteins. The pH buffering capacity of myofibrillar proteins significantly decreased from 3.14 ± 0.03 mM H+/(mL × ΔpH) down to 2.55 ± 0.03 mM H+/(mL × ΔpH) after oxidation with 50 mM H2O2. Both oxidized myofibrillar proteins and histidine showed a high pH buffering capacity at pH near 5.8, which is the histidine pKa value. Here, we hypothesize that oxidation-induced changes in the pH buffering capacity of myofibrillar proteins were driven by oxidative modification of histidine and structural changes of myofibrillar proteins. The significance of this study to food industry may be the awareness that protein oxidation may affect pH through changes in buffering capacity. And the use of antioxidants, especially those targeting at histidine will be promising in addressing this issue.

Effects of calcium chloride on the gelling and digestive characteristics of myofibrillar protein in Litopenaeus vannamei.

In this study, the effects of CaCl2 (0, 25, 50, 75, and 100 mM) on the gelling and digestive properties of the myofibrillar protein (MP) in Litopenaeus vannamei were investigated. The results showed that increasing CaCl2 concentration led to changes in the tertiary structure of MP. Specifically, compared with the control group, a 64.31 % increase in surface hydrophobicity and a 45.90 % decrease in the sulfhydryl group were observed after 100 mM CaCl2 treatment. Correspondingly, the water holding capacity and strength of the MP gel increased by 24.46 % and 55.99 %, respectively. These changes were positively correlated with the rheological properties, microstructure pore size, and content of non-flowable water. The mechanical properties of MP gel were improved, and the microstructure became more compact with the increase in CaCl2 concentration. Furthermore, the particle size of the digested MP gels decreased in the presence of CaCl2, which improved the digestion characteristics of MP gels.

Improved emulsifying properties of water-soluble myofibrillar proteins at acidic pH conditions: Emphasizing pH-regulated electrostatic interactions with chitosan.

Water-soluble muscle protein with enhanced functionalities has attracted great interest for low-salt food design. Electrostatic interactions of chitosan (CS) with myofibrillar proteins (MP) in water-aqueous solution at acidic pHs (4.0-6.5) were investigated, and how pH regulated complex formation, microstructures, conformation changes, and emulsifying capacity was systematically explored. At pH 4.0-4.5, MP and CS were positively charged and displayed a co-soluble system, exhibiting small particles and high solubility. When the pH increased to near the isoelectric point (pI) of MP (pH 5.0-6.0), electrostatic interactions largely inhibited the aggregation of MP by forming smaller particle complexes. The flexible structures and improved amphiphilic properties promoted protein absorption at the oil-water interface, further improving the emulsion stability. When the pH increased to 6.5, large aggregates were formed causing poor functionalities. This study could provide great insights to further exploit meat-protein-based low-salt functional foods in novel food design.

Study on the interaction and gel properties of pork myofibrillar protein with konjac polysaccharides.

BACKGROUND: Natural myofibrillar protein (MP) is sensitive to changes in the microenvironment, such as pH and ionic strength, and therefore can adversely affect the final quality of meat products. The aim of this study was to modify natural MP as well as to improve its functional properties. Therefore, the quality improvement effect of konjac polysaccharides with different concentrations (0, 1.5, 3, 4.5 and 6 g kg-1 protein) on MP gels was investigated. RESULTS: With a concentration of konjac polysaccharides of 6 g kg-1 protein, the composite gel obtained exhibited a significant improvement of water binding (water holding capacity increased by 7.71%) and textural performance (strength increased from 29.12 to 37.55 N mm, an increase of 8.43 N mm). Meanwhile, konjac polysaccharides could help to form more disulfide bonds and non-disulfide covalent bonds, which enhanced the crosslinking of MP and maintained the MP gel network structure. Then, with the preservation of α-helix structure (a significant increase of 8.11%), slower protein aggregation and formation of small aggregates, this supported the formation of a fine and homogeneous network structure and allowed a reduction in water mobility. CONCLUSION: During the heating process, konjac polysaccharides could absorb the surrounding water and fill the gel system, which resulted in an increase in the water content of the gel network and enhanced the gel-forming ability of the gel. Meanwhile, konjac polysaccharides might inhibit irregular aggregation of proteins and promote the formation of small aggregates, which in turn form a homogeneous and continuous gel matrix by orderly arrangement. © 2023 Society of Chemical Industry.

Combined effect of ultrasound treatment and κ-carrageenan addition on the enhancement of gelling properties and rheological behavior of myofibrillar protein: An underlying mechanisms study.

This work aimed to investigate the combined effect of ultrasound (US) treatment and κ-carrageenan (KC) addition on the gelling properties and rheological behaviors of myofibrillar protein (MP). Without US treatment, the KC incorporation promoted the gel strength and water-holding capacity (WHC) of MP gels. These properties were further improved by 20 min US treatment with gel strength of 98.61 g and WHC of 79.87 %, which was mainly attributed to changes associated with hydrophobic interactions and disulfide bonds and the transformation from α-helix to ß-sheet in MP gels. In addition, US treatment for 20 min effectively resulted in a more homogeneous polymer distribution of the MP-KC mixed system, leading to lower particle size and the largest G' and G″ values of the MP-KC mixed gels. However, longer US treatment times (30, 40 and 50 min) rendered lower gel strength, WHC, storage modulus and loss modulus of MP-KC mixed gels, which was mainly due to the formation of loose and disordered gel structures. Our present results indicated that the application of US to MP for an intermediate treatment time (20 min) combined with KC provides a potential and novel strategy to promote the gel qualities of heat-induced MP gels.

Low-voltage electrostatic field enhances the frozen force of -12 ℃ to suppress oxidative denaturation of the lamb protein during the subsequent frozen storage process after finishing initial freezing.

The effect of low-voltage electrostatic field (LVEF) assisted -9 °C (LVEF-9) and -12 °C (LVEF-12) frozen, non-LVEF-assisted -9 °C (NLVEF-9) and -12 °C (NLVEF-12) frozen, and conventional frozen (CF-18, -18 °C) storage on the muscle microstructure and the oxidative denaturation of the lamb protein during the subsequent frozen storage process after finishing initial freezing was investigated. Compared with NLVEF-9, LVEF-9, and NLVEF-12, LVEF-12 maintained the better integrity of muscle microstructure, demonstrated by smaller holes, more complete Z-line and M-line, and no significant difference with CF-18 (P > 0.05). Furthermore, LVEF-12 effectively inhibited protein oxidative denaturation as shown by the lower carbonyl content, surface hydrophobicity, and higher total/active sulfhydryl groups and Ca2+-ATPase activity. Moreover, LVEF-12 effectively maintained the integrity of the secondary and tertiary structure of proteins, reduced cross-linking aggregation of proteins, and sustained better functional properties, as shown by higher α-helix content, fluorescence intensity, protein solubility, and lower R-value, disulfide bonds.

Excessive free radical grafting interferes with the macromolecular association and crystallization of brined porcine myofibrils during heat-set gelatinization.

Free radical grafting and oxidative modification show superiority in myofibrillar protein (MP) aggregation patterns during salting process, but their consequent formation mechanisms of protein hydration network require further evaluation. Herein, we explored the effect of salt-curing (0, 1, 3 and 5 %) on MP protein polymer substrate, water-protein interaction, crystallization events and thermal stability under H2O2/ascorbate-based hydroxyl radical (•OH)-generating system (HRGS) (1, 10, 20 mM H2O2). Results showed that moderate salting (≤3%) favored the water binding of MP gels during the oxidation course. Accordingly, the maximum thermal stability (Tm) of MP gels was obtained at 3 % salting could be greatly attributed to the protein chain solubilization and refolding process. However, 5 % salt synergized with •OH oxidation intensified diffraction peak 2 (the most striking diffraction feature). Microstructural analysis validated a maximum compactness of MP gel following brining with 5 % salt at potent oxidation strength (20 mM H2O2). This study maybe promises efficient strategy to the myogenetic fibril products and biomaterials.

Mechanism on the Synergistic Gelation of the Myofibrillar Protein Composite Gel Enhanced by "Clean-Label" Skin Functional Protein Powders.

The concept of healthiness and sustainability has promoted the innovation and development of "clean-label" products. Herein, this study aims to explore the influence mechanism of "clean label" skin protein powder (FPP) on the gelation properties of myofibrillar proteins (MPs). Specifically, the addition of FPP (0.2-4.0%) can improve the water holding capability and texture properties of MP composite gels. When the FPP concentration is over 1.0%, the composite gels exhibit no significant water loss during centrifugation. Dynamic rheology and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis results revealed that FPP can slow the aggregation and denaturation of myosin and promote the formation of disulfide bonds between myofibril proteins, thus forming a stable network structure. Structural observation revealed that FPP can fill into the MP gel and lead to the formation of compact gel structures. Besides, with the increase of FPP concentration, the chemical forces involved in structural stabilization change significantly. Specifically, hydrophobic interaction and hydrogen bonding are the dominant forces at a lower FPP concentration (0.2 and 0.4%), while the ionic bond and disulfide bond are the dominant forces at a higher concentration. Overall, this work demonstrated that FPP can significantly improve the gel functionality of MP by altering the gel structure and strengthening the molecular forces.

Structure determination and analysis of titin A-band fibronectin type III domains provides insights for disease-linked variants and protein oligomerisation.

Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.

Lysine Methyltransferase SMYD1 Regulates Myogenesis via skNAC Methylation.

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

Monosaccharide-induced glycation enhances gelation and physicochemical properties of myofibrillar protein from oyster (Crassostrea gigas).

Glycation offers a promising potential to improve protein gelling properties in food industries. Therefore, the study was aimed to illustrate the effect of five monosaccharides (erythrose-aldotetrose, xylose-aldopentose, glucose-aldohexose, galactose-aldohexose, and fructose-ketohexose) with different carbon numbers and structure on the structure-gelling relationship of myofibrillar protein (MP) from oyster (Crassostrea gigas). Results showed that monosaccharides significantly increased the glycation degree of MP by increasing sulfhydryl content, forming stable tertiary conformation and decreasing surface hydrophobicity. Moreover, the gel properties of MP like gel strength, water holding capacity, water mobility were improved by alleviating aggregation including the increase of solubility and the decrease of particle sizes. Oyster MP glycated by glucose (aldohexose) possessed the optimal gel properties. Molecular docking simulation showed that hydrogen bonds and hydrocarbon bonds were the mainly non-covalent binding modes. The study will provide a theoretical basis for oyster protein glycation and expand its application on food gel.

Regulation mechanism of curcumin-loaded oil on the emulsification and gelation properties of myofibrillar protein: Emphasizing the dose-response of curcumin.

The regulation mechanism of curcumin (CUR) in the oil phase on the emulsification and gelation properties of myofibrillar protein (MP) was investigated. CUR enhanced the emulsifying activity index (EAI) of MP but decreased its turbiscan stability index (TSI) and surface hydrophobicity, which exacerbated oil droplet aggregation. Medium amounts (200 mg/L) of CUR changed the 3D network architectures of emulsion gels from lamellar to reticular, improving the gels' water-holding capacity (WHC), storage modulus, springiness, and cohesiveness. Besides, the LF-NMR revealed that CUR had limited effects on the mobility of immobilized and free water. The α-helix of MP in gels with medium amounts of CUR decreased from 51% to 45%, but the ß-sheet increased from 23% to 27% compared to those without CUR. Overall, CUR has the potential to become a novel structural modifier in emulsified meat products due to its dose-response.