Random Coil Research Articles

Preliminary In silico Analysis of Adenylate Kinase 1 (ADK1) of Echinococcus granulosus as a Candidate for Vaccination against Cystic Echinococcosis

Background: A neglected zoonosis, Cystic Echinococcosis (CE), is most common in developing nations worldwide. Vaccination is, therefore, helpful in preventing the disease. Objective: Predicting the main biochemical properties of E. granulosus Adenylate Kinase 1 (ADK1) and its possible B-cell and T-cell-binding epitopes as a valuable candidate for immunization was the goal of the current study. Methods: Predictions were made to determine biochemical, antigenic, structural, and subcellular characteristics, along with the immunogenic epitopes, using several online servers. Results: The extracellular 22 KDa protein had no allergenicity, while it possessed hydrophilicity (GRAVY: -0.286), stability (instability: 17.48), tolerance to a wide range of temperatures (aliphatic: 93.45), and 17 post-translational modification sites. The secondary structure mostly comprised helices and random coils and the 3D model was generated using Robetta server (confidence: 0.88). Common B-cell epitopes were discovered by three servers and screened for antigenic, allergenic, and solubility traits. Moreover, MHC-associated epitopes for mice and humans were predicted in E. granulosus ADK1 with subsequent screening. Conclusion: This work offers a foundation for further investigation on designing an effective vaccination against CE. Further empirical research study with the examined protein solely or combined with other antigens is needed.

From Enzyme to Vaccine: Preliminary Insights into the Immunogenic Epitopes in Adenylate Kinase 8 (ADK8) of Echinococcus granulosus

Background: Globally, developing livestock countries are affected by Cystic Echinococcosis (CE), a neglected zoonosis. Thus, vaccination would help to prevent this neglected zoonosis. Objective: This study aimed to predict the primary biochemical characteristics of E. granulosus Adenylate Kinase 8 (ADK8) and its potential B- and T-cell-binding epitopes as a candidate for vaccination. Methods: Prediction were conducted on multiple web servers to identify immunogenic epitopes as well as biochemical, antigenic, structural, and subcellular properties. Results: The extracellular 38.78 KDa protein possessed no allergenic fragments, while it showed hydrophilicity (GRAVY: -0.239), wide thermotolerance (aliphatic: 98.02), and 36 post-translational modification sites. The secondary structure mostly included helices and random coils, and the best-generated 3D model by the Robetta server had a confidence score of 0.79. Three servers identified common B-cell epitopes and evaluated them for solubility, allergenicity, and antigenic properties. Furthermore, following the screening, MHC-associated epitopes for humans and mice were predicted in the E. granulosus ADK8. Conclusion: These results provide a basis for future research into the development of a proper vaccine against CE. Additional empirical studies using the protein and/or its epitopes alone or in combination with additional antigens are demanded.

Static magnetic field effects on the secondary structure and elasticity of collagen molecules; a possible biophysical approach to treat keratoconus

Type I collagen is among the major extracellular proteins that play a significant role in the maintenance of the cornea's structural integrity and is essential in cell adhesion, differentiation, growth, and integrity. Here, we investigated the effect of 300 mT Static Magnetic Field (300 mT SMF) on the structure and molecular properties of acid-solubilized collagens (ASC) isolated from the rat tail tendon. The SMF effects at molecular and atomic levels were investigated by various biophysical approaches like Circular Dichroism Spectropolarimetery (CD), Fourier Transform Infrared Spectroscopy (FTIR), Zetasizer light Scattering, and Rheological assay. Exposure of isolated type I collagen to 300 mT SMF retained its triple helix. The elasticity of collagen molecules and the keratoconus (KCN) cornea treated with SMF decreased significantly after 5 min and slightly after 10, 15, and 20 min of treatments. The exposure to 300 mT SMF shifted the Amid I bond random coil to antiparallel wave number from 1647 to 1631 cm−1. The pH of the 300 mT SMF treated collagen solution increased by about 25 %. The treatment of the KCN corneas with 300 mT SMF decreased their elasticity significantly. The promising results of the effects of 300 mT SMF on the collagen molecules and KCN cornea propose a novel biophysical approach capable of manipulating the collagen's elasticity, surface charges, electrostatic interactions, cross binding, network formation and fine structure. Therefore, SMF treatment may be considered as a novel non-invasive, direct, non-chemical and fast therapeutic and manipulative means to treat KCN cornea where the deviated physico-chemical status of collagen molecules cause deformation.

Comprehensive bioinformatics assessments of the ROP34 of Toxoplasma gondii to approach vaccine candidates

IntroductionRhoptries proteins (ROPs) are crucial throughout different stages of the Toxoplasma gondii (T. gondii) lifecycle, playing key roles in both the invasion of host cells and their subsequent survival. ROP34 is particularly noteworthy as it significantly influences host gene expression and aids in the transition from the tachyzoite to the bradyzoite form.Materials and methodsThis research utilized various bioinformatics tools to assess physico-chemical properties, allergenic and antigenic characteristics, sites for post-translational modifications (PTMs) and protein's secondary and three-dimensional structures of the ROP34 protein. Furthermore, the study identified potential B-cell, MHC-binding, and cytotoxic T-lymphocyte (CTL) epitopes within the ROP34 sequence.ResultsThe ROP34 peptide comprised 553 amino acid residues, with a calculated average molecular weight (MW) of 61.60149 kDa, an aliphatic index of 73.98, and a GRAVY score of − 0.554. The antigenicity of the multi-epitope peptide was estimated to be 0.526563 and 0.6025 by the ANTIGENpro and VaxiJen servers, respectively, suggesting ROP34 as an immunogenic protein with no allergenic potential. Secondary structure analysis revealed a composition of 52.80% random coil, 36.17% alpha helix, and 11.03% extended strand. The Ramachandran plot for the refined model depicted that 97.46% of the residues were situated in the favored region.ConclusionThis in silico research serves as a foundation for designing effective immunization tactics to target toxoplasmosis. The present article lays the groundwork for future studies and offers perspectives for the advancement of an appropriate toxoplasmosis vaccine.Graphical

Formation of thermal-induced microgels from soy protein hydrolysates: Effects of selective proteolysis on glycinin/β-conglycinin

This study explored the impact of selective proteolysis on the formation of thermally induced soy protein microgels. Glycinin hydrolysate (GH) and β-conglycinin hydrolysate (CH) were obtained by subjecting soy protein isolate to selective proteolysis for different hydrolysis time (10–90 min), as confirmed by SDS-PAGE. In the early stages of hydrolysis, free sulfhydryl, surface hydrophobicity, storage modulus (G′) and loss modulus (G″) of GH and CH increased, which enhanced their gelling potential. However, as hydrolysis time increased, the gel properties of the hydrolysates progressively weakened. Structural characterization of microgels revealed that GH yielded microgels with smaller particle sizes and coarser and relatively dispersed granular structures, while CH resulted in microgels with lower potential values, smoother surfaces, and lumps resembling strand-like formations. Analysis of the structure and intermolecular force of microgels showed that the microgel formed by the GH gradually tended to be disordered, whereas the secondary structure of microgels formed by CH showed lower random coil content, resulting in a dense gel network aggregated through disulfide bonding, hydrophobic interactions and hydrogen bonding as demonstrated by frequency-dependent storage moduli measurements. Overall, this study presents a thorough characterization of microgels and shows that they can be tailored by selective proteolysis, which enables controlling the β-conglycinin/glycinin ratio of soy protein.

Effects of High-Pressure Homogenization on the Structure and Functional Properties of Solenaia oleivora Proteins.

Solenaia oleivora, a rare freshwater shellfish with high protein quality, is unique to China. However, the poor hydrosolubility and functional properties of Solenaia oleivora proteins hinder their utilization in food products. Herein, the alkaline dissolution-isoelectric precipitation method was used for the extraction of Solenaia oleivora proteins. Furthermore, the impact of high-pressure homogenization (HPH) treatment varying from 0 to 100 MPa on the structure and functional properties of Solenaia oleivora proteins was investigated. The obtained results indicated that HPH treatment decreased the α-helix content and enhanced the β-sheet and random coil content. Furthermore, the HPH caused the unfolding of protein structure, exposing aromatic amino acids, increasing the free thiol group content, and enhancing surface hydrophobicity. As the homogenization pressure increased from 0 to 100 MPa, the particle size of Solenaia oleivora proteins decreased from 899 to 197 nm with the polymer dispersity index (PDI) value decreased from 0.418 to 0.151, the ζ-potential increased from -22.82 to -43.26 mV, and the solubility increased from 9.54% to 89.96%. Owing to the significant changes in protein structure and solubility, the emulsifying, foaming, and digestive properties of Solenaia oleivora proteins have been significantly improved after treatment with HPH.

Anti-solvent precipitation for encapsulation of oyster protein hydrolysate nanoparticles: Effect on off-flavor elimination and bioaccessibility

In this study, to investigate the potential for delivering bioactive substances, various cereal proteoglycan nanoparticles were prepared using anti-solvent precipitation for the delivery of oyster protein hydrolysate (OPH). The hydrogen bonding and hydrophobic interaction were the driving forces for OPH embedding. OPH-Loaded Zein/NaCas/Gum Arabic composite nanoparticles could self-assemble into spherical (191 nm) particles with strong stability, uniform distribution (about PDI = 0.2), and high negative charge (about −28 mV). The secondary structure of OPH-Loaded zein was converted from α-helix to random coil with heat treatment. The off-flavor, including (E, E)-2,4-heptadienal, octadecane, and 1-octanol in OPH-Loaded Zein nanoparticles significantly decreased. Furthermore, these nanoparticles containing OPH had excellent oxidation resistance, in vitro digestibility, and non-toxicity in Caco-2 and HT-29 cells. Overall, this work provides an efficient strategy for OPH delivery, suggesting that OPH could be ingested as a nutraceutical in powder form while also promoting the development of high-flavor-quality oyster products.

Influence of Initial Secondary Structure on Conformation and Mechanical Properties of Spider Silk Protein Gels.

Recombinant spider silk protein (RSP) is a promising biomaterial for developing high-performance materials independent of fossil fuels. In this study, we investigated the influence of the initial secondary structure of RSPs on the properties of RSP-based hydrogels. By altering the initial structure of RSP to β-sheets (β-RSP), α-helices (α-RSP), and random coils (rc-RSP) through solvent treatment, we compared the structures and mechanical properties of the resulting gels. Solid-state NMR revealed a β-sheet-rich structure in all gels, with the α-RSP gel exhibiting significantly higher strength and Young's modulus compared to the rc-RSP gel. X-ray diffraction revealed that the α-RSP gel had a unique crystalline structure, distinguishing it from the β-RSP and rc-RSP gels. The different initial secondary structures possibly lead to variations in the crystalline and network structures of the molecular chains within the gels, explaining the superior mechanical properties observed in the α-RSP gels.

Monomer Composition as a Mechanism to Control the Self-Assembly of Diblock Oligomeric Peptide-Polymer Amphiphiles.

Diblock oligomeric peptide-polymer amphiphiles (PPAs) are biohybrid materials that offer versatile functionality by integrating the sequence-dependent properties of peptides with the synthetic versatility of polymers. Despite their potential as biocompatible materials, the rational design of PPAs for assembly into multichain nanoparticles remains challenging due to the complex intra- and intermolecular interactions emanating from the polymer and peptide segments. To systematically explore the impact of monomer composition on nanoparticle assembly, PPAs were synthesized with a random coil peptide (XTEN2) and oligomeric alkyl acrylates with different side chains: ethyl, tert-butyl, n-butyl, and cyclohexyl. Experimental characterization using electron and atomic force microscopies demonstrated that the tail hydrophobicity impacted accessible morphologies. Moreover, the characterization of different assembly protocols (i.e., bath sonication and thermal annealing) revealed that certain tail compositions provide access to kinetically trapped assemblies. All-atom molecular dynamics simulations of micelle formation unveiled key interactions and differences in core hydration, dictating the PPA assembly behavior. These findings highlight the complexity of PPA assembly dynamics and serve as valuable benchmarks to guide the design of PPAs for a variety of applications, including catalysis, mineralization, targeted sequestration, antimicrobial activity, and cargo transportation.

Underlying mechanisms of different polysaccharides on the texture characteristics and protein digestibility of soybean curd

This study aimed to investigate the effects of the addition of potato starch (PS) and guar gum (GG) at different concentrations (0.4 and 0.8%) on the physical properties, protein conformation, and in vitro digestibility of soybean curd. The results indicated that the PS acted as a filler in the protein structure, enhancing the firmness, springiness, and chewiness of the soybean curd. GG encapsulated the disorganized filamentous protein matrix, increasing the adhesiveness of the soybean curd. The addition of GG and PS reduced the hydrophobic interactions and disulfide bonds responsible for cross-linking soybean curd proteins, while forming more hydrogen bonds with the proteins (p < 0.05). GG formed the most hydrogen bonds with the proteins, as well as reduced β-sheet content and transformed into more random coil and β-turn (p < 0.05). Also, the addition of GG (particularly at 0.8%) significantly reduced the degree of protein hydrolysis (31.98%) in soybean curd, resulting in fewer small molecular weight peptides and lower total amino acid content (3386.79 μg/g protein) at the end of in vitro digestion (p < 0.05). In contrast, the addition of a small amount of PS (0.4%) did not affect the protein digestibility of soybean curd. This study clarified the impact of polysaccharide additions on modifying the soybean curd texture and protein digestibility.

Cryo-EM and solid state NMR together provide a more comprehensive structural investigation of protein fibrils.

The tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure.

The Genome-Wide Identification of the Dihydroflavonol 4-Reductase (DFR) Gene Family and Its Expression Analysis in Different Fruit Coloring Stages of Strawberry.

Dihydroflavonol 4-reductase (DFR) significantly influences the modification of flower color. To explore the role of DFR in the synthesis of strawberry anthocyanins, in this study, we downloaded the CDS sequences of the DFR gene family from the Arabidopsis genome database TAIR; the DFR family of forest strawberry was compared; then, a functional domain screen was performed using NCBI; the selected strawberry DFR genes were analyzed; and the expression characteristics of the family members were studied by qRT-PCR. The results showed that there are 57 members of the DFR gene family in strawberry, which are mainly expressed in the cytoplasm and chloroplast; most of them are hydrophilic proteins; and the secondary structure of the protein is mainly composed of α-helices and random coils. The analysis revealed that FvDFR genes mostly contain light, hormone, abiotic stress, and meristem response elements. From the results of the qRT-PCR analysis, the relative expression of each member of the FvDFR gene was significantly different, which was expressed throughout the process of fruit coloring. Most genes had the highest expression levels in the full coloring stage (S4). The expression of FvDFR30, FvDFR54, and FvDFR56 during the S4 period was 8, 2.4, and 2.4 times higher than during the S1 period, indicating that the DFR gene plays a key role in regulating the fruit coloration of strawberry. In the strawberry genome, 57 members of the strawberry DFR gene family were identified. The higher the DFR gene expression, the higher the anthocyanin content, and the DFR gene may be the key gene in anthocyanin synthesis. Collectively, the DFR gene is closely related to fruit coloring, which lays a foundation for further exploring the function of the DFR gene family.

Structural analyses of apricot pectin polysaccharides

Apricot pectin polysaccharides' fine structure was performed using HPSEC, HPAEC-PAD, GC–MS, NMR and FTIR spectroscopies. Purified pectin fraction (F1AP) was composed of D-galacturonic acid, L-rhamnose, D-arabinose and D-galactose, Mw ∼ 1588 kDa. F1AP was eluted by water and with 0.2 M NaCl from DEAE Sepharose fraction resulting in two distinct fractions, F1AP1 and F1AP6, with different structures, molecular weights, and conformations, providing insights into their structural diversity. F1AP1 neutral properties were related to its association with protein. F1AP1 had a backbone of (1 → 4)-linked-D-galacturonic acid and (1 → 2)-linked-L-rhamnopyranosyl residues branched with arabinogalactan including multiple glycosidic linkages of T-α-Araf, 3-α-Araf, 5-α-Araf, T-α-Arap, 2-α-Arap, t-Galp, 2-Galp, 3-Galp, 4-Galp, 6-Galp, 2,4-Galp, 3,4-Galp, 3,6-Galp and 4,6-Galp side chains, having methyl and acetylated groups, and a high molecular weight (1945 kDa). The Mark-Houwink exponent was 0.276, indicating a compact spherical conformation. While the other F1AP6 fraction consists predominately of less methylated HG regions of pectin polysaccharides. The molar mass of this fraction was 117.5 kDa, which adopted a stiffer and random coil conformation. This knowledge allows us to evaluate how the balance of chemical structure and physical properties of the two pectin domains may manifest itself in the isolated structure of apricot pectin and its applications.

Covalent interaction of ovalbumin with proanthocyanidins improves its thermal stability and antioxidant and emulsifying activity.

The structure of proanthocyanidins (PC) contains a large number of active phenolic hydroxyl groups, which makes it have strong antioxidant capacity. This study investigated the structural and functional properties of ovalbumin (OVA) modified by its interaction with PC. It was found that on increasing the concentration ratio of PC to OVA from 10:1 to 40:1, the free amino and total sulfhydryl contents of OVA decreased from 470.59 ± 38.77 and 29.81 ± 0.31 nmol mg-1 to 96.61 ± 4.55 and 21.22 ± 0.78 nmol mg-1, respectively, and the free sulfhydryl content increased from 7.65 ± 0.41 to 9.48 ± 0.58 nmol mg-1. These results indicated that CN and CS bonds were formed and PC was covalently linked with OVA. The PC content in the OVA-PC conjugates increased from 281.93 ± 12.92 to 828.81 ± 46.09 nmol mg-1 on increasing the concentration ratio of PC to OVA from 10:1 to 40:1. The contents of α-helix and β-turn of OVA decreased, and the contents of β-sheet and random coil increased, confirmed by circular dichroism. The tertiary structure of OVA was also altered according to the results of fluorescence and ultraviolet absorption spectra. The surface hydrophobicity of OVA-PC conjugates decreased with increasing bound polyphenol content. The conjugation of OVA to PC significantly improved its emulsification and antioxidant activity and denaturation temperature. This study may provide valuable information for improving OVA's functional properties and its PC conjugates for applications in the food industry. © 2024 Society of Chemical Industry.

Dynamic mechanism and structural property analysis of microfluidic-obtained pea protein fiber

Although protein-based fibers have been developed, the preparation of inexpensive, safe and environmentally friendly plant protein fibers is still a hot topic. Herein, we constructed the microfluidic chip to produce protein fiber using pea protein and a small amount of cellulose nanofibers. The results demonstrated that compared to pea protein alone, the pea protein-based fiber produced through microfluidic spinning exhibited a smooth and dense surface structure, good thermal stability, higher Zeta potential and antioxidant activity, and lower surface hydrophobicity and sensitization. Furthermore, we comprehensively revealed the dynamic formation mechanism of pea protein-based fiber. During the fiber preparation process, hydrodynamics led the protein and cellulose nanofibers to align gradually along the flow direction, enhancing their molecular orientation. Additionally, strong hydrodynamic shear force altered the protein structure, transitioning its spatial structure from random coil and α-helix to β-sheet, and promoting a binding of protein to cellulose nanofibers. This research may offer theoretical guidance for the application of pea protein-based fiber in the preparation of hypoallergenic protein-based fibers and the construction of foodborne delivery materials.

Investigating the change mechanism and quantitative analysis of minced pork gel quality with different starches using Raman spectroscopy

Minced pork gel often faces challenges during preparation, such as sticking and splitting, which can result in a loose structure, significantly compromises its quality. This study aimed to improve minced pork gel quality by incorporating different starches: mung bean starch (MBS), potato starch (POS), pea starch (PES), and corn starch (CS), focusing on enhancing water-holding capacity (WHC) and gel strength. Raman spectroscopy was employed to investigate the mechanisms behind gel quality changes and to develop quantitative predictions of WHC and gel strength in response to starch addition. The results showed that 15% starch addition was optimal, with POS performing best. Notably, starch addition promoted α-helix and random coil transformation into β-sheet and β-turn in protein structure. Additionally, the starch-minced pork gel system exhibited enhanced cross-linking and density through hydrophobic interactions, contributing to improved WHC and gel strength. Finally, to quantitatively assess the gel quality change under different starches, support vector machine (SVM), uninformative variable elimination-SVM (UVE-SVM), genetic algorithm-SVM (GA-SVM), and adaptive reweighted sampling-SVM (CARS-SVM) modeling were built. Among these, UVE-SVM model achieved the optimal performance for both WHC (Rp2 = 0.8553, RPD = 2.1567) and gel strength (Rp2 = 0.8508, RPD = 2.1981). Therefore, this study demonstrates that the addition of starch, particularly POS, can enhance minced pork gel quality by improving both WHC and gel strength. Moreover, it establishes Raman spectroscopy coupled with UVE-SVM as a reliable method for non-destructive, and rapid detection of these quality parameters in minced pork gel under different starch conditions.

Influence of fractions with different molecular weight distributions from high-amylose starches on their digestibility after recrystallization

Type 3 resistant starches (RS3) were prepared from debranched starch (DBS) with different average degree of polymerization (DP) generated from high-amylose pea starch (HAPS) and high-amylose maize starch (HAMS). The results showed that RS3 with DP 35 and DP 39 had the highest RS content (74.5 % and 75.0 %, respectively) after cooking, which were remarkably higher than those of RS3 prepared from mixed fractions (60.6 % and 49.0 %, respectively) and other separated fractions (34.1–63.0 %). The multi-scale structures of RS3, including short-range molecular order, crystalline structure, micro-ordered aggregate structure, microscopic structure, and particle size distribution, were influenced by the average DP. Notably, RS content was positively correlated with the proportion of DP 51–80 and negatively correlated with the proportion of DP 21–30. DBS with DP 51–80 contributed to a more organized micro-ordered aggregate structure at nanometer scale and a larger aggregate structure at micrometer scale, which improved the resistance of RS3 to amylolytic enzymes. However, DBS with DP 21–30 tended to form random coil structure that were more easily to be digested. This research offered new insights into the structure-digestibility relationship of RS3, which is meaningful for the development of RS3 with high resistance to amylolytic enzymes after cooking.

Effects of papain hydrolysate of Pseudosciaena crocea roe on the physical and oxidative stability of water-in-oil emulsions

This study investigates the effects of hydrolysates from Pseudosciaena crocea roe on the physical and oxidative stability of oil-in-water emulsions, emphasizing the structure-activity relationship between peptide structure and emulsifying properties. The results showed that, compared to other proteases, hydrolysates prepared with papain (PCRHs) at moderate degrees of hydrolysis (DH 4.68% and 6.8%) exhibited superior emulsifying activity index (EAI) and emulsifying stability index (ESI). PCRHs with DH of 4.68%, 6.8% formed smaller particle sizes (89.8 ± 3.52 nm, 112 ± 2.65 nm) and more stable emulsions. EAI and ESI of PCRHs were significantly negatively correlated with emulsion particle size and significantly positively correlated with surface hydrophobicity, amino acid composition, relative molecular mass, random coil content and total sulfhydryl groups. Additionally, although PCRHs with DH of 4.68% produced smaller and more stable emulsion particles, PCRHs with DH of 6.8% significantly inhibited primary and secondary lipid oxidation products formation. Correlation analysis revealed that emulsions with smaller particle sizes exhibited poorer oxidative stability, and secondary volatile oxidation products better reflected the oxidative stability of emulsions. Therefore, hydrolysates from Pseudosciaena crocea roe protein with moderate hydrolysis, particularly PCRHs with DH of 6.8%, demonstrate potential as natural emulsifiers to maintain the physical and oxidative stability of oil-in-water emulsions.

Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis.

This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2-14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4-8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens.

Fabrication and characterization of whey protein isolate-tryptophan nanoparticles by pH-shifting combined with heat treatment

L-Tryptophan (Trp) is an essential amino acid with numerous health benefits. However, incorporating Trp into food products is limited due to its pronounced bitter taste. Encapsulating Trp in nanoparticles by using other natural biopolymers is a potential strategy to mask the bitter taste of Trp in the final products. Whey protein isolate (WPI), composed of alpha-lactalbumin (α-LA), bovine serum albumin (BSA), and beta-lactoglobulin (β-LG), has played a crucial role in delivering bioactive compounds. In order to incorporate Trp within WPI, the present study used a combination of pH-shifting andthermal treatment to fabricatewhey protein isolate-tryptophan nanoparticles (WPI-Trp-NPs). During the pH-shifting technique, WPI unfolds at high pH, such as pH 11, and the dissociated WPI molecules are refolded when pH is shifted back to neutral, creating particles with uniform dispersion and encapsulating smaller particles surrounding them in solution. Further, the well-distributed nanoparticles formed by pH-shifting might encourage the formation of more uniform nanoparticles during subsequent thermal treatment. TheWPI-Trp particles have an average particle size of 110.1 nm and a low average PDI of 0.20. Fluorescence spectroscopy confirmed the encapsulation of Trp by WPI, which shows higher fluorescence when the Trp is encapsulated by the WPI. Surface hydrophobicity, circular dichroism, particle size, free sulfhydryl, and antioxidant activity were used to characterize the WPI-Trp-NPs. WPI-Trp-NPs formed by pH-shifting combined with heating showed a higher surface hydrophobicity and free sulfhydryl content than the untreated WPI-Trp mixture. The conversion of α-helix into random coil in the WPI secondary structure indicated a more disordered structure of the modified whey protein. Molecular docking results indicate the interactions between Trp and WPI, including alpha-lactalbumin (α-LA), bovine serum albumin, and beta-lactoglobulin (β-LG), were mainly driven by hydrophobic interactions and hydrogen bonding. The binding affinity between Trp and these proteins was ranked as α-LA>BSA>β-LG. The combination of pH-shifting and heating improved the functionalityof WPI and was an effective way to fabricate WPI-Trp nanoparticles.