Search Results (366)

The bifunctional glutathione synthase (GshF) is able to catalyze glutathione synthesis and is favored for industrial application due to its lack of product inhibition. However, its practical use is limited by moderate catalytic efficiency and poor thermostability. Here, we applied ancestral sequence reconstruction (ASR) to engineer a more robust ancestral GshF (Anc427) with thermal denaturation temperature of 56.2 ± 0.2 °C, representing an increase of 10.8 ± 0.2 °C over the probe enzyme (St-GshF). Additionally, Anc427 exhibited a thermal half-life (t_1/2) of 3465.7 min at 40 °C, representing a 20-fold increase over that of St-GshF. Under optimal conditions (pH 7.0, 37 °C), Anc427 displayed a specific activity of 3.3 ± 0.02 U·mg⁻¹, representing a 20% enhancement compared to St-GshF. Structural modeling and molecular dynamics simulations indicated that the improved stability can be attributed to increased structural rigidity in Anc427. These findings demonstrate that ASR effectively enhances both thermostability and catalytic activity of GshF, significantly advancing its potential for industrial biocatalysis. Full article

The specific heat capacity of skinned muscle in an adhering rigor solution was studied with differential scanning calorimetry (DSC) heating runs to search for a heat sink in the sarcomere of the muscle. To elucidate the contribution of major myoproteins to heat capacity, myosin and actin were partially removed by high-KCl and gelsolin treatments, respectively. Differential heat denaturation of myosin (together with α-actinin) and actin was induced to confirm their contributions. On the DSC curve, aside from the endothermic peaks representing ice melting and protein denaturation, the steady baseline level showed a significant increase in basal heat capacity in the presence of skinned muscle compared to the rigor solution alone. In the physiological temperature range from 10 to 25 °C, untreated skinned muscle in the native state (non-denatured) introduced an extra basal heat capacity of 0.4 J K⁻¹ (g evaporable weight)⁻¹, which was diminished by both removing and denaturing actin and was additionally increased by removing myosin; myosin denaturation had little effect on the basal heat capacity. Based on these results, we considered actin to be the fundamental source of extra basal heat capacity, which was partly suppressed by the thermally stable region of myosin under rigor conditions. This extra basal heat capacity was roughly preserved at sub-zero temperatures, suggesting the involvement of non-freezing water molecules. The extra basal heat capacity may have contributed to thermal buffering during muscle function via actin-associated hydration. As a supplemental result, we found a small reversible endothermic peak around −21 °C, which was suppressed in the presence of skinned muscle. Heating beyond the denaturing temperatures reduced this suppression effect. Full article

Protein-based materials such as human serum albumin (HSA) have demonstrated significant potential for the development of novel wound management materials. For the first time, the formation of HSA-based hydrogels was proposed using a combination of thermal- and ethanol-induced approaches. The combination of phosphate-buffered saline (PBS) and limited (up to 20% v/v) ethanol content offers a promising strategy for fabricating human serum albumin-based hydrogels with tunable properties. The hydrogel formation was studied using in situ dynamic light scattering (DLS) for qualitative and semi-quantitative analysis of the patterns of protein hydrogel formation through thermally induced gelation. The rheological properties of human serum albumin-based hydrogels were investigated. Hydrogels synthesized via thermally induced gelation using a denaturing agent exhibit a dynamic viscosity ranging from 100 to 10,000 mPa·s. The biocompatibility, biodegradability, and structural stability of human serum albumin-based hydrogels were comprehensively evaluated in physiologically relevant media. These human serum albumin-based hydrogels represent a promising platform for developing topical therapeutic agents for wound management and tissue engineering applications. This study investigated the kinetics of tetracycline release from human serum albumin-based hydrogels in PBS and fetal bovine serum (FBS). All tested formulations of HSA-based hydrogels loaded with tetracycline (1 mg/mL) demonstrated antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Corynebacterium striatum strains. Full article

This study investigated the technological, nutritional, and sensory effects of incorporating soybean flour and radish leaves into steamed manti, with emphasis on moisture-loss kinetics, protein denaturation, true retention (TR), and relative nutrient density (RND). Four formulations were examined: potato control (PC), potato + soy (PS), greens control (GC), and greens + soy (GS). Steaming induced compositional increases in dry matter, ash, protein, and fat due to moisture reduction rather than absolute changes in solids. Greens-based formulations exhibited significantly lower moisture-loss and protein-denaturation rate constants, indicating stronger hydration stability and structural resistance during thermal processing. These kinetic advantages translated into higher TR values for protein and fat in GC and GS compared with potato-based samples. Soy flour substantially increased protein and lipid content and improved dough cohesiveness but did not influence thermal behavior or moisture-loss kinetics within the same matrix. When nutrient delivery was normalized to energy content, soy- and greens-enriched manti showed the highest RND values, reflecting a favorable combination of nutrient retention and lower caloric density. Sensory evaluation confirmed that soy enhanced textural attributes, while radish leaves contributed desirable juiciness and aroma. Overall, the combined use of radish leaves and soybean flour offers a sustainable approach to producing nutrient-dense, sensory-acceptable traditional foods while supporting the valorisation of leafy by-products. Full article

Eel (Anguilla) is an aquatic animal with high nutritional value and multiple health benefits for the human body. To fully utilize its processing by-products fish bone, this study optimized the enzymatic preparation process of using BP neural network and GA genetic algorithm, with collagen extraction yield as the key evaluation metric, and characterized the properties of the obtained collagen. The results demonstrated that the optimal extraction conditions for eel bone collagen were as follows: enzyme dosage of 2%, hydrolysis time of 2.65 h, solid-to-liquid ratio of 1:22, and ultrasonic pretreatment for 21 min at 250 W power, achieving an extraction yield of 57.6%. The main amino acids identified were glycine, glutamic acid, proline, and arginine. SDS-PAGE electrophoresis revealed that eel bone collagen exhibited structural characteristics of type I collagen. Raman spectroscopy and X-ray diffraction indicated an intact triple-helix structure with partial ordered features. The DSC and TGA results demonstrated good thermal stability, with a denaturation temperature of 106.73 °C. SEM imaging displayed a loose, porous fibrous network structure, while rheological analysis suggested potential biomedical material properties. The findings of this study provide fundamental data for the high-value utilization and development of eel bone resources. Full article

Royal jelly, a high-value natural product rich in bioactive compounds, is highly susceptible to quality deterioration during storage and processing. However, current quality standards rely predominantly on basic physicochemical parameters and measuring the content of 10-hydroxy-2-decenoic acid (10-HDA), which fail to capture the comprehensive and dynamic nature of its freshness. This significant knowledge gap hinders the accurate assessment, prediction, and control of royal jelly quality throughout its supply chain. To address this limitation, this review systematically elucidates the molecular mechanisms underlying the deterioration of royal jelly freshness, including key pathways such as protein denaturation, Maillard reactions, enzymatic inactivation, and lipid oxidation, and analyzes the combined effects of intrinsic and extrinsic factors on its quality stability. It highlights the potential applications of novel biochemical markers—including major royal jelly proteins (MRJPs), Maillard reaction products, enzymatic activity indicators, and energy metabolites—while comparing the advantages and limitations of traditional chromatographic techniques with modern rapid sensing and spectroscopic analysis methods. Regarding preservation, a critical yet inadequately summarized area, this review systematically evaluates the applicability and limitations of various approaches, including low-temperature storage, drying treatments, non-thermal sterilization, microencapsulation, and modified atmosphere packaging. Future directions for integrated quality control are outlined, providing a theoretical basis for holistic quality management of royal jelly. Full article

This study systematically compared ultrasound-assisted extraction (UAE) with five other methods (hot water extraction (HWE), microwave-assisted extraction (MAE), acid extraction (FTACP), alkali extraction (FTAIP), and enzyme-assisted extraction (EAE)) for their effects on the yield, physicochemical properties, and bioactivities of Rosa laevigata Michx. polysaccharides. The results demonstrated UAE’s superior performance: it achieved a higher polysaccharide yield (31.27%) than HWE, FTACP, and FTAIP, approaching that of MAE and EAE. SEM observation revealed that UAE-derived polysaccharides exhibited a uniform porous network with smooth surfaces and excellent dispersibility, outperforming the irregular aggregates or structural loosening observed in other methods. Notably, UAE polysaccharides showed remarkable cholesterol-binding capacity (31.18 mg/g) and FRAP reducing power (0.0423 mmol/g), which highlights their potential for functional food applications. Structural analyses (FT-IR, XRD, TGA) confirmed that UAE better preserved the native conformation and thermal stability of polysaccharides, whereas chemical (FTACP/FTAIP) and high-temperature (MAE) methods induced molecular degradation. In conclusion, UAE, as an eco-friendly and low-denaturation technique, offers an optimal strategy for the high-value utilization of R. laevigata polysaccharides. Full article

This study examines the effects of extrusion cooking on the biofunctional, rheological, thermal, and structural properties of corn starch (CS)/whey protein isolate (WPI) blends (100/0, 50/50, 0/100 w/w, both raw and extruded) during in vitro gastrointestinal digestion. Extrusion and in vitro digestion increased antioxidant activity (2,2′-Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) Diammonium Salt and 2,2-Diphenyl-1-Picrylhydrazyl). Extrusion improved the bioaccessibility of angiotensin-converting enzyme (ACE-1) inhibitory peptides, leading to high inhibition (>90%) in the intestinal phase across all samples, with this effect consistent between raw and extruded samples during digestion. The in vitro digestion process changes the rheological behavior of the samples, from a non-Newtonian fluid (dilatant) to a Newtonian fluid. Notably, extruded CS maintained pseudoplastic behavior across all phases. Thermally, extrusion resulted in complete gelatinization of CS and denaturation of WPI, as evidenced by the absence of endotherms. Structurally, extrusion induced unfolding of WPI α-helix and β-sheet regions, leading to the formation of β-turns and random coils, which could enhance enzyme accessibility. For CS, a decrease in the degree of double helix and order was observed, indicating an alteration of its ordered molecular structure. Additionally, the extrusion process slightly increased the amount of resistant starch. This work shows that extrusion generates antioxidant compounds by bioactive peptide release. Full article

Heat-induced β-carotene synthesis in Dunaliella salina typically compromises biomass accumulation, resulting in a biomass–β-carotene trade-off. This study demonstrates that exogenous putrescine (Put) alleviates this conflict through temperature-dependent mechanisms. At 28 °C (optimal for growth), 10⁻⁶ M Put increased biomass by 9.52% and β-carotene yield by 10.72%, probably by accelerating electron transport and relatively mitigating the loss of photosynthetic function. At 34 °C (optimal for β-carotene synthesis), 10⁻⁷ M Put enhanced biomass by 9.68% and β-carotene yield by 35.71% through a process associated with nitric oxide (NO) accumulation, involving antioxidant synergy and controlled reactive oxygen species (ROS) signaling, which activated photoprotective carotenogenesis. At 40 °C (extreme thermal stress), 10⁻⁷ M Put maintained β-carotene levels 44.99% above the control despite a 2.50% biomass reduction, reflecting a shift toward photoprotection via elevated non-photochemical quenching (NPQ) and sustained electron transport beyond photosystem II (δ_RO). Put’s hierarchical modulation of redox homeostasis, photosystem plasticity, and NO signaling underpinned its temperature-dependent efficacy. Peak NO levels correlated with β-carotene yield, while thermodynamic enzyme denaturation at 40 °C limited protection. These findings establish a temperature–concentration framework for Put application that alleviates the biomass–β-carotene trade-off under climate variability. Full article

The growing demand for sustainable proteins has driven interest in Limnospira platensis (Spirulina) due to its high protein content. However, the presence of the cell wall limits the availability and recovery of proteins within it. Conventional alkaline extraction is widely applied but often results in low yields and excessive solvent consumption. This study compares the efficiency and functional properties of Spirulina proteins extracted using an alkaline method and high-pressure homogenisation (HPH) at 20, 50, 80 and 100 MPa. Following isoelectric precipitation, proteins were collected in precipitate and supernatant fractions and characterized for yield, solubility, phycobiliproteins content, emulsifying and foaming properties, water– and oil–holding capacity, thermal stability and rheological behaviour. Microscopy confirmed progressive cell disruption with increasing homogenization pressures. HPH at 50 MPa increased protein extraction by 28% compared to alkaline extraction and significantly (p < 0.05) improved solubility, oil-holding capacity, foaming and emulsion properties. Phycobiliproteins, particularly C–phycocyanin, were more efficiently recovered in HPH supernatants, achieving a higher purity index than the alkaline method. Rheological analysis showed weak gel-like network formation, whereas excessive mechanical stress reduced functionality. Overall, HPH emerges as an interesting method for obtaining Spirulina proteins with enhanced technological properties; however, pressure optimisation is required to avoid denaturation and functionality loss. Full article

Background/Objectives: Natural plant-based compounds, especially black pepper extract, are known to have anti-inflammatory, antibacterial, and antioxidant qualities that promote procollagen formation and wound healing. This study focused on developing a collagen-based composite enriched with P. nigrum extract in powder form, designed to enhance the efficacy of the antibiotic cefazolin while promoting the healing of chronic wounds. Methods: The polyphenolic P. nigrum extract was obtained by ultrasound-assisted extraction and was characterised by UHPLC-MS/MS and spectrophotometry. Antimicrobial and antioxidant activities were assessed using conventional methods. Pharmacokinetic and pharmacodynamic parameters were evaluated for the specific taxon compounds using Deep-RK. P. nigrum extract was incorporated into a collagen hydrogel with arginine and freeze-dried. The powders were characterised by FTIR, SEM, TGA-DSC, and DLS. The antimicrobial activity and potential synergistic effects with cefazolin were evaluated on reference microbial strains and isolates from infected wounds. Biocompatibility and hemocompatibility were evaluated, as well as wound closure in vitro. Results: Polyphenols, including phenolic acids, stilbenes, anthocyanins, and flavonoids, which provide a potent antioxidant capacity through electron transfer mechanisms (FRAP, CUPRAC), were abundant in the P. nigrum extract. FTIR and SEM analyses confirmed the integration of phenolic compounds into the collagen–arginine matrix without protein denaturation. TGA–DSC data showed thermal stabilisation at moderate extract concentrations. The extract exhibited predominantly bacteriostatic antibacterial activity and antibiofilm effects, with synergy/additivity with cefazolin, especially at medium doses. Tests on keratinocytes confirmed biocompatibility, and hemocompatibility demonstrated an excellent safety profile, with protection against AAPH-induced oxidative stress. Conclusions: Overall, collagen powders with P. nigrum extract at moderate/low concentrations combine stability, antibiotic-enhanced activity, and cellular compatibility, making them promising adjuvants for the topical treatment of chronically infected wounds. Full article

We here present a novel and sensitive methodology for determining the melting point (MP) of Bovine Serum Albumin (BSA) from micromolar to picomolar concentration levels under label-free conditions. At 1 pM we could model the melting with a sharp Gaussian. However, from the transient state observed during the melting process by using a simple exponential decay model, we determined a time constant of 67 s. We applied this methodology by studying a 3.3 pM sample of a botulinum toxin A (BoNT-A) (stabilized with 2.8 nanomolar denatured Human Serum Albumin (HSA)). We were able to determine the Tm of BoNT-A in the presence of approximately 1000-fold more concentrated HSA. This method enables the detection of protein melting transitions at picomolar concentrations without the use of a fluorescence dye. Its sensitivity and simplicity make it a valuable analytical tool for studying protein stability in diluted pharmaceutical formulations. This method is useful for correlating thermal conformational changes with catalytic function. Full article

To investigate the effects of different treatment methods on soybean protein amyloid fibrils (SPAF), this study examined the effects of ultrasonication, cold plasma, heat, and NaCl treatment on the formation, structure, and functional properties of SPAF. SPAF structural analyses indicated that all treatments promoted SPAF assembly, with the order of effectiveness being: heat treatment > ultrasonication > cold plasma treatment > NaCl treatment. Regarding functional properties, the heat treatment group also demonstrated superior overall performance, including the highest solubility (88.15%), optimal emulsifying activity (79.63 m²/g) and foaming capacity (169.44%), and the highest thermal denaturation temperature (107.49 °C). Conversely, ultrasonication and cold plasma treatments, which generated shorter fibrils, offered moderate functional improvements. In contrast, NaCl treatment provided limited functional enhancement due to the formation of coarse aggregates. Consequently, heat treatment was identified as the most effective approach to promote SPAF formation and enhance functional properties. These findings provide a theoretical basis for the process optimization of SPAF in the food industry. Full article

RNA triple helices are relatively understudied, including their interactions with small molecules. In this study, we evaluated eight previously reported triplex-binding molecules (TBMs) for their functional effects on the premature and mature MALAT1 triple helix. Based on UV thermal denaturation experiments, the TBMs berberine, coralyne, sanguinarine, berenil, and neomycin selectively stabilize the Hoogsteen interface of the MALAT1 triple helix. Moreover, fisetin, luteolin, and quercetin were more sensitive to nucleotide composition, whereas berberine, coralyne, sanguinarine, and berenil were more sensitive to changes in the length of the major-groove triple helix. Most TBMs could not outcompete MALAT1 triple helix-binding proteins, except for neomycin. Surface plasmon resonance experiments demonstrated that berberine and sanguinarine display relatively quick association and dissociation binding profiles. Treating human colorectal carcinoma cells with each of the TBMs reduced MALAT1 levels by ~20–60%. This study demonstrates that TBMs broadly recognize the premature and mature MALAT1 triple helix but exhibit subtle sensitivities, suggesting that TBMs can be designed to selectively bind triple helices based on nucleotide composition, length, and structural context. Full article

Novel nanocomposites based on carbon black or multi-walled carbon nanotubes functionalized with carboxylic groups and Neutral red electropolymerized from reline were obtained in a one-step protocol and used for DNA biosensor development. The synthesis was carried out in potentiodynamic mode in a deep eutectic solvent reline consisting of a mixture of choline chloride and urea. The nanocomposite based on carbon black and poly(Neutral red) was applied for a voltammetric DNA biosensor developed to discriminate DNA damage. The sensor developed allowed the native, thermally denatured, and chemically oxidized DNA discrimination with either current changes or peak potential shifts. The nature of the DNA used had affected the sensor’s analytical response value. The DNA biosensor suggested was tested for the assessment of antioxidant capacity in such beverages as tea, coffee, white wine, and fruit-based drink purchased from local market. Simple, fast, and inexpensive approach of sensor modifying layer assembly would be demanded in control of food products and beverages quality, as well as for medical purposes. Full article