Protein Lysozyme Research Articles

Extraction of Lysozyme from Chicken Albumen Using Polyelectrolyte Complexes

Cells use droplet-like membrane-less organelles (MLOs) to compartmentalize and selectively take-up molecules, such as proteins, from their internal environment. These membraneless organelles can be mimicked by polyelectrolyte complexes (PECs) consisting of oppositely charged polyelectrolytes. Previous research has demonstrated that protein uptake strongly depends on the PEC composition. This suggests that PECs can be used to selectively extract proteins from a multi-protein mixture. With this in mind, the partitioning of the protein lysozyme in four PEC systems consisting of different weak and strong polyelectrolyte combinations is investigated. All systems show similar trends in lysozyme partitioning as a function of the complex composition. The release of lysozyme from complexes at their optimal lysozyme uptake composition is investigated by increasing the salt concentration to 500mm NaCl or lowering the pH from 7 to 4. Complexes of poly(allylamine hydrochloride) and poly(acrylic acid) have the best uptake and release properties. These are used for selective extraction of lysozyme from a hen-egg white protein matrix. The (back)-extracted lysozyme retains its enzymatic activity, showing the capability of PECs to function as extraction media for proteins.

Fabrication of protein-coated titanium dioxide nanoparticles for cellular uptake fluorescence imaging and treatment of colorectal cancer

Titanium dioxide (TiO2) coated with different proteins has exhibited exceptional bio-compatibility, leading to various biomedical engineering applications. With the use of green and chemical reduction methods, we fabricated Titanium dioxide nanoparticles that were protein-coated. Bovine serum albumin (BSA), lysozyme proteins, zein, and collagen have been used to coat titanium dioxide-aryl nanoparticles of the form TiO2-NPs. However, in both cases, no catalysts or other stabilizing agents were used. These images of TiO2-NPs fabricated using the green method show high crystallinity. It is a malignant colorectal tumour with dysfunctional cellular processes that cause colorectal cancer cells. It is hoped that studies employing SW1417 cells would give mechanistic ideas on the specifics of the amplification in cancers. This was done by flow cytometry utilizing and laser confocal fluorescence microscopy (LCFM) on the SW1417 colorectal cell line. Of the protein-coated Titanium dioxide nanoparticles fabricated green methods, BSA@TiO2-NPs were the most readily absorbed. Of all TiO2-NPs, lysozyme@TiO2-NPs fabricated by the chemical reduction technique were the most effectively internalized by SW1417 cells out of TiO2-NPs types. However, TiO2-NPs fabricated by the green methodology were coated with zein and lysozyme and tiny. A hydrophobic covering is also on the two nanoparticles. There is a possibility that the variation in hydrophobicity and charge affected the internalization process. Colorectal diagnostic and therapeutic compounds might be synthesized from those coated nanoparticles that were effectively internalized.

Effects of Dietary Mannan Oligosaccharides on Non-Specific Immunity, Intestinal Health, and Antibiotic Resistance Genes in Pacific White Shrimp Litopenaeus vannamei.

This study was conducted to comprehensively investigate the beneficial effects of a mannan oligosaccharide product (hereinafter called MOS) on Litopenaeus vannamei and optimum level of MOS. Five isonitrogenous and isolipid diets were formulated by adding 0%, 0.02%, 0.04%, 0.08%, and 0.16% MOS in the basal diet. Each diet was randomly fed to one group with four replicates of shrimp in an 8-week feeding trial. The results showed that dietary MOS improved the growth performance and the ability of digestion of shrimp. Dietary MOS significantly increased the activity of total superoxide dismutase, catalase, and glutathione peroxidase and decreased the content of malondialdehyde in plasma of shrimp. Dietary MOS significantly increased the activity of alkaline phosphatase and lysozyme in plasma and the hemocyte counts. Dietary MOS significantly upregulated the expression of Toll, lysozyme, anti-lipopolysaccharide factor, Crustin, and heat shock protein 70 in the hepatopancreas. And dietary MOS significantly upregulated the expression of intestinal mucin-2, mucin-5B, and mucin-19, while it decreased the expression of intestinal mucin-1 and macrophage migration inhibitory factor. Dietary MOS improved the bacterial diversity; increased the abundance of Lactobacillus, Bifidobacterium, Blautia, and Pseudoalteromonas; and decreased the abundance of Vibrio in the intestine. Shrimp fed MOS diets showed lower mortality after being challenged by Vibrio parahaemolyticus. Notably, this study found a decrease in antibiotic resistance genes and mobile genetic elements after MOS supplementation for the first time. The present results showed that diet with MOS supplementation enhanced the organismal antioxidant capacity and immunity, improved intestinal immunity, optimized intestinal microecology, mitigated the degree of antibiotic resistance, and increased the resistance to V. parahaemolyticus in L. vannamei, especially when supplemented at 0.08% and 0.16%.

Low Fouling Polysulfobetaines with Variable Hydrophobic Content.

Amphiphilic polymer coatings combining hydrophilic elements, in particular zwitterionic groups, and hydrophobic elements comprise a promising strategy to decrease biofouling. However, the influence of the content of the hydrophobic component in zwitterionic coatings on the interfacial molecular reorganization dynamics and the anti-fouling performance is not well understood. Therefore, coatings of amphiphilic copolymers of sulfobetaine methacrylate 3-[N-2'-(methacryloyloxy)ethyl-N,N-dimethyl]-ammonio propane-1-sulfonate (SPE) are prepared which contain increasing amounts of hydrophobic n-butyl methacrylate (BMA). Their fouling resistance is compared to that of their homopolymers PSPE and PBMA. The photo-crosslinked coatings form hydrogel films with a hydrophilic surface. Fouling by the proteins fibrinogen and lysozyme as well as by the diatom Navicula perminuta and the green algae Ulva linza is assessed in laboratory assays. While biofouling is strongly reduced by all zwitterionic coatings, the best fouling resistance is obtained for the amphiphilic copolymers. Also in preliminary field tests, the anti-fouling performance of the amphiphilic copolymer films is superior to that of both homopolymers. When the coatings are exposed to a marine environment, the reduced susceptibility to silt incorporation, in particular compared to the most hydrophilic polyzwitterion PSPE, likely contributes to the improved fouling resistance.

Zwitterionic Peptides Reduce Accumulation of Marine and Freshwater Biofilm Formers.

Zwitterionic peptides are facile low-fouling compounds for environmental applications as they are biocompatible and fully biodegradable as their degradation products are just amino acids. Here, a set of histidine (H) and glutamic acid (E), as well as lysine (K) and glutamic acid (E) based peptide sequences with zwitterionic properties were synthesized. Both oligopeptides (KE)4K and (HE)4H were synthesized in d and l configurations to test their ability to resist the nonspecific adsorption of the proteins lysozyme and fibrinogen. The coatings were additionally tested against the attachment of the marine organisms Navicula perminuta and Cobetia marina as well as the freshwater bacterium Pseudomonas fluorescens on the developed coatings. While the peptides containing lysine performed better in protein resistance assays and against freshwater bacteria, the sequences containing histidine were generally more resistant against marine organisms. The contribution of amino acid-intrinsic properties such as side chain pKa values and hydrophobicity, as well as external parameters such as pH and salinity of fresh water and seawater on the resistance of the coatings is discussed. In this way, a detailed picture emerges as to which zwitterionic sequences show advantages in future generations of biocompatible, sustainable, and nontoxic fouling release coatings.

Droplet-Based Evaporative System for the Estimation of Protein Crystallization Kinetics.

Crystallization is a potential cost-effective alternative to chromatography for the purification of biotherapeutic proteins. Crystallization kinetics are required for the design and control of such processes, but only a limited quantity of proteins is available during the initial stage of process development. This article describes the design of a droplet-based evaporative system for the evaluation of candidate crystallization conditions and the estimation of kinetics using only a droplet (on the order of μL) of protein solution. The temperature and humidity of air fed to a flow cell containing the droplet are controlled for evaporation and rehydration of the droplet, which are used for manipulating supersaturation. Dual-angle images of the droplet are taken and analyzed on-line to obtain the droplet volume and crystal sizes. Crystallization kinetics are estimated based on a first-principles process model and experimental data. Tight control of temperature and humidity of the air, fast and accurate image analysis, and accurate estimation of crystallization kinetics are experimentally demonstrated for a model protein lysozyme. The estimated kinetics are suitable for the model-based design and control of protein crystallization processes.

Comparison of the Antimicrobial and Hemolytic Activities of Various Forms of Silver (Ions, Nanoparticles, Bioconjugates) Stabilized in a Zeolite Matrix.

A quantitative and qualitative comparison of the antimicrobial and hemolytic activities of silver in various states, in the form of ions, nanoparticles, and bioconjugates with the antimicrobial protein lysozyme stabilized in an inert zeolite matrix, has been carried out. A synthetic zeolite with a β structure was chosen as a zeolite matrix. Using the ion-exchange method, the method of chemical reduction, and treating the matrix with a silver hydrosol with specified characteristics, samples of zeolites with the same silver content in various forms (Ag+, Ag° - Ag°/Lyz) in the amounts of 0.8 and 5 wt % have been synthesized. The samples obtained were studied by a complex of physicochemical research methods: X-ray diffraction, UV absorption spectroscopy, low-temperature nitrogen adsorption, electron microscopy, and atomic absorption. Antimicrobial activity was assessed against antibiotic-resistant Gram-negative microbe (e.g., Escherichia coli ML-35, Pseudomonas aeruginosa 522/17 MDR, Klebsiella pneumoniae ESBL 344) and Gram-positive microbe (e.g., Staphylococcus aureus 1399/17). The hemolytic activity in relation to human erythrocytes was estimated. The results obtained showed significant antimicrobial activity with a simultaneously high hemolytic activity of ionic silver. Silver nanoparticles have a lower level of antimicrobial activity and toxicity. Bioconjugates of silver nanoparticles and lysozyme showed an optimal combination of antimicrobial properties and lack of hemolytic activity.

Water-soluble pillar[5]arene sulfo-derivatives self-assemble into biocompatible nanosystems to stabilize therapeutic proteins

Pillar[5]arenes containing sulfonate fragments have been shown to form supramolecular complexes with therapeutic proteins to facilitate targeted transport with an increased duration of action and enhanced bioavailability. Regioselective synthesis was used to obtain a water-soluble pillar[5]arene containing the fluorescent label FITC and nine sulfoethoxy fragments. The pillar[5]arene formed complexes with the therapeutic proteins binase, bleomycin, and lysozyme in a 1:2 ratio as demonstrated by UV–vis and fluorescence spectroscopy. The formation of stable spherical nanosized macrocycle/binase complexes with an average particle size of 200 nm was established by dynamic light scattering and transmission electron microscopy. Flow cytometry demonstrated the ability of macrocycle/binase complexes to penetrate into tumor cells where they exhibited significant cytotoxicity towards A549 cells at 10−5–10−6 M while maintaining the enzymatic activity of binase.

Functional-State Dependence of Picosecond Protein Dynamics.

We examine temperature-dependent picosecond dynamics of two benchmarking proteins lysozyme and cytochrome c using temperature-dependent terahertz permittivity measurements. We find that a double Arrhenius temperature dependence with activation energies E1 ∼ 0.1 kJ/mol and E2 ∼ 10 kJ/mol fits the folded and ligand-free state response. The higher activation energy is consistent with the so-called protein dynamical transition associated with beta relaxations at the solvent-protein interface. The lower activation energy is consistent with correlated structural motions. When the structure is removed by denaturing, the lower-activation-energy process is no longer present. Additionally, the lower-activation-energy process is diminished with ligand binding but not for changes in the internal oxidation state. We suggest that the lower-energy activation process is associated with collective structural motions that are no longer accessible with denaturing or binding.

The Effect of Feed Supplementation with EM Bokashi® Multimicrobial Probiotic Preparation on Selected Parameters of Sow Colostrum and Milk as Indicators of the Specific and Nonspecific Immune Response

The aim of the study was to determine the effect of EM Bokashi® on selected parameters of the specific and nonspecific immune response of sows by in colostrum and milk samples. The percentage of cells with expression of CD19+, CD5+CD19+, CD21+, SWC3a (macrophage/monocyte), and CD11b+ molecules on the monocytes and granulocytes as well as the concentrations of lysozyme and acute phase proteins — serum amyloid-A (SAA) and haptoglobin (Hp) were evaluated. The study was carried out on a commercial pig farm, including 150 sows (Polish Large White × Polish Landrace) at the age of 2–4 years. Sixty female sows were divided into two groups: I — control and II — experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® in the amount of 10 kg/tonne of feed was added to the basal feed from mating to weaning. The material for the study consisted of colostrum and milk. The samples were collected from all sows at 0, 24, 48, 72, 96, 120, 144, and 168 h after parturition. The study showed that exposure of the pregnant sow to the probiotic microbes contained in EM Bokashi® significantly affects the immunological quality of the colostrum and milk and caused an increase in the percentage of the subpopulations of B cells with CD19+, CD21+, and CD5+CD19+ expression in the colostrum and milk, which demonstrates an increase in the protective potential of colostrum and indicates stimulation of humoral immune mechanisms that protect the sow and the piglets against infections.

Highly sensitive and selective lateral flow aptasensor for anti-coagulant dabigatran etexilate determination in blood

Dabigatran etexilate (DBG) is a new anticoagulant drug (commercially sold under the names Pradaxa® and Pradax™) that replaces Warfarin, the landmark agent for anticoagulation therapy. Inadequate administration of DBG or in the cases of massive bleeding that occurs after renal impairment, DBG therapy can carry a substantial life-threatening risks. One of the major limitations of DBG treatment is the lack of a simple and quick tool for measuring its level in blood in the case of massive bleedings or emergency operations. In this work, we have incorporated a previously isolated aptamer for DBG to develop a simple competitive lateral flow aptasensor (LFA) for the determination of DBG in buffer and blood samples. A full-length 60-mer aptamer as well as a truncated 38-mer aptamer were conjugated to gold nanoparticles (AuNPs) via thiol-Au coupling chemistry. After appropriate AuNP surface passivation steps, the aptamer's core region was hybridized with 8-mer biotinylated sequences. The conjugated particles could be capture on the test line by the interaction of the biotin molecules with a previously deposited streptavidin. Incubation of the conjugated particles with DBG causes the aptamer to undergo a conformational change that releases the 8-mer biotinylated sequences and result in the disappearance of the test line. Lysozyme protein was used to construct the control line that non-specifically interacts with the conjugated particles whether or not the target compound is present. The developed LFA achieves 20 nM detection level in buffer and blood samples, operates within the nanomolar range, and shows excellent selectivity against potential interfering molecules. The developed sensor could help assessing the levels of DBG in medical conditions that require rapid interventions.

Naringenin Regulates Lipopolysaccharide-Induced Abnormal Airway Surface Liquid Secretion

Airway surface liquid (ASL) is one of the key factors affecting the respiratory system's physiological function. Abnormal ASL secretion can increase the incidence of various respiratory diseases. Lipopolysaccharide (LPS) stimulation can damage the airway epithelial barrier, affect the concentration of ASL contents, and down-regulate ion channel expression, which in turn causes abnormal ASL secretion. Naringenin, which exists in many Citrus foods, has the ability to promote airway surface liquid secretion. This work is designed to investigate the regulatory mechanism of naringenin on LPS-induced abnormal ASL secretion. The effects of naringenin and LPS on the viability of Calu-3 cells were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS). ASL secretion volume was measured by a micropipette on air–liquid interface cultured cells. The concentration of Cl−, Na+, lysozyme, and total protein in ASL were respectively measured by assay kits. The mRNA expressions were determined by quantitative real-time polymerase chain reaction, and proteins were measured by enzyme-linked immunosorbent assay. The results indicated that LPS could affect ASL secretion and regulate cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 1 (AQP1) and aquaporin 5 (AQP5) expression. Naringenin had the ability to regulate the ASL secretion by increasing secretion volume, and Cl− and Na+ concentrations, reducing lysozyme and total protein content, and regulating CFTR, AQP1, and AQP5 expression. This study indicated that naringenin had regulating effects to attenuate LPS-induced abnormal ASL secretion.

The effect of synbiotics and probiotics on the growth performance, gastrointestinal function and health status of turkeys

ABSTRACT The aim of this study was to evaluate the growth performance, gastrointestinal function and health status of turkeys fed diets supplemented with synbiotic preparations, as compared with commercial probiotic feed additives. The experiment lasted for 15 weeks. The research material comprised 600 female BIG 6 turkeys (6 treatments, 5 replicates, 20 birds per replicate). The turkeys from the control group (I) received a diet without additives. Groups II and III received a basal diet with the addition of probiotic BioPlus 2B or Cylactin at 0.4 g/kg diet, respectively. In groups IV, V and VI turkeys were fed diets with synbiotic preparations S1 (L. reuteri, L. plantarum, L. pentosus, S. cerevisiae + inulin), S2 (L. reuteri, L. plantarum, L. pentosus, S. cerevisiae, L. rhamnosus + inulin) and S3 (L. reuteri, L. plantarum, L. pentosus, S. cerevisiae, L. rhamnosus, L. paracasei + inulin) at 0.5 g/kg diet, respectively. The following parameters were monitored: growth performance, carcass quality, the chemical composition of meat, the structure (length, weight, villus height, crypt depth) and functional parameters (pH, viscosity) of selected segments of the gastrointestinal tract, and the health status of birds (lysozyme, gamma-globulins, ceruloplasmin and total protein). Dietary supplementation with probiotics and synbiotics contributed to an increase in the final body weights of turkeys, a decrease in the feed conversion ratio and an increase in values of the European Production Efficiency Factor (p ≤ 0.05). Synbiotics improved the immune status of birds by increasing serum gamma-globulin levels and decreasing ceruloplasmin activity at 8th week of age (p ≤ 0.05). Synbiotics and probiotics also contributed to a decrease in crop and caecal pH (p ≤ 0.05). The analysed additives had no effect on carcass dressing percentage, carcass quality characteristics or the chemical composition of breast muscles. The tested synbiotics as well as commercial probiotics can be valuable feed additives, improving the growth performance and immune status of turkeys.

Molecular insights into α-synuclein interaction with individual human core histones, linker histone, and dsDNA.

α-Synuclein (αS) plays a key role in Parkinson's disease (PD). The αS nuclear role, its binding affinity and specificity to histones and dsDNA remains unknown. Here, we have measured the binding affinity ( ) between αS wild-type (wt) and PD-specific αS S129-phosphorylation mimicking (S129E) mutant with full-length and flexible tail truncated individual core histones (H2a, H2b, H3, and H4), linker histone (H1), and carried out αS-dsDNA interaction studies. This study revealed that αS(wt) interacts specifically with N-terminal flexible tails of histone H3, H4, and flexible tails of H1. The αS(S129E) mutant recognizes histones similar to αS(wt) but binds with higher affinity. Intriguingly, αS(S129E) showed a binding affinity for control proteins (bovine serum albumin and lysozyme), while no interaction was seen for αS(wt). Based on our above observation, we contemplate that the physio-chemical properties of αS with S129-phosphorylation has changed compared to αS(wt), resulting in interaction for other proteins, which is the basis for Lewy body formation. Besides, this study showed αS binding to dsDNA is weak and nonspecific. Overall, αS specificity for histone binding suggests that its nuclear role is possibly driven through histone interaction.

How Do Proteins Associate with Nanoscale Metal-Organic Framework Surfaces?

It is well known that colloidal nanomaterials, upon exposure to a complex biological medium, acquire biomolecules on their surface to form coronas. Porous nanomaterials present an opportunity to sequester biomolecules and/or control their orientation at the surface. In this report, a metal-organic framework (MOF) shell around gold nanorods was compared to MOF nanocrystals as potential protein sponges to adsorb several common proteins (lysozyme, beta-lactoglobulin-A, and bovine serum albumin) and potentially control their orientation at the surface. Even after correction for surface area, MOF shell/gold nanorod materials adsorbed more protein than the analogous nanoMOFs. For the set of proteins and nanomaterials in this study, all protein-surface interactions were exothermic, as judged by isothermal titration calorimetry. Protein display at the surfaces was determined from limited proteolysis experiments, and it was found that protein orientation was dependent both on the nature of the nanoparticle surface and on the nature of the protein, with lysozyme and beta-lactoglobulin-A showing distinct molecular positioning.

Anti-fouling and protein separation of PVDF-g-PMAA@MnO2 filtration membrane with in-situ grown MnO2 nanorods

MnO2 nanorods with controllable scale were grown in the PVDF-g-PMAA modified membrane to form PVDF-g-PMAA@ MnO2 membrane through the in situ redox reaction of KMnO4 solution, which is confirmed by scanning electron microscopy (SEM) and X-ray energy-dispersion spectroscopy (EDX). The pore size of the membrane decreased with the increase of KMnO4 solution concentration. The thermodynamic stability and the hydrophilicity of the membrane were also enhanced by the MnO2 nanorods. The water flux, bovine serum albumin (BSA)/Lysozyme protein solution flux and rejection, flux recovery, etc. showed effective improvement of the anti-fouling performance of the PVDF-g-PMAA@ MnO2 membrane. More importantly, it can effectively separate BSA from lysozyme, which provided a potential application in the field of biology, food, and other industrial fields for the requirement of separation and purification.

In Situ Forming iEDDA Hydrogels with Tunable Gelation Time Release High-Molecular Weight Proteins in a Controlled Manner over an Extended Time.

Off-target interactions between reactive hydrogel moieties and drug cargo as well as slow reaction kinetics and the absence of controlled protein release over an extended period of time are major drawbacks of chemically cross-linked hydrogels for biomedical applications. In this study, the inverse electron demand Diels-Alder (iEDDA) reaction between norbornene- and tetrazine-functionalized eight-armed poly(ethylene glycol) (PEG) macromonomers was used to overcome these obstacles. Oscillatory shear experiments revealed that the gel point of a 15% (w/v) eight-armed PEG hydrogel with a molecular weight of 10 kDa was less than 15 s, suggesting the potential for fast in situ gelation. However, the high-speed reaction kinetics result in a risk of premature gel formation that complicates the injection process. Therefore, we investigated the effect of polymer concentration, temperature, and chemical structure on the gelation time. The cross-linking reaction was further characterized regarding bioorthogonality. Only 11% of the model protein lysozyme was found to be PEGylated by the iEDDA reaction, whereas 51% interacted with the classical Diels-Alder reaction. After determination of the mesh size, fluorescein isothiocyanate-dextran was used to examine the release behavior of the hydrogels. When glucose oxidase was embedded into 15% (w/v) hydrogels, a controlled release over more than 250 days was achieved. Overall, the PEG-based hydrogels cross-linked via the fast iEDDA reaction represent a promising material for the long-term administration of biologics.

Evaluation of Utilizing Functionalized Graphene Oxide Nanoribbons as Compatible Biomaterial for Lysozyme.

Graphene oxide nanoribbons with superior physicochemical properties acquired from graphene and carbon nanotubes have been used in various applications including biomedical applications. For biomedical applications, it is of utmost importance to understand how these graphene oxide nanoribbons interact with proteins and the influence they have on protein conformation and function. In this regard, an attempt has been made to evaluate the utility of graphene oxide nanoribbons as a compatible biomaterial for lysozyme (Lys) protein. In this study, graphene oxide nanoribbons (GONRs) synthesized from multiwalled carbon nanotubes (MWCNTs) were first functionalized with (3-aminopropyl)triethoxysilane (APTES) and further modified with vanillin (Val) to obtain Val-APTES-GONRs. On characterization, it was found that the Val-APTES-GONRs material had a ribbonlike morphology with abundant functionalities for interaction with protein. On evaluation of Val-APTES-GONRs as a compatible biomaterial for Lys, studies revealed that a lower concentration of the as-synthesized material has less influence on the conformation and the structure of Lys with better activity, whereas higher concentrations of the as-synthesized material had a greater influence on conformation and the structure of Lys with decreased activity. Overall, the thermal stability of Lys was maintained after introducing the Val-APTES-GONRs material. In addition, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) and Raman spectroscopies were performed for Lys composites with Val-APTES-GONRs for further understanding biomolecular interactions. This study is beneficial for designing advanced graphene-based materials for numerous bioinspired applications and better biomaterials for biotechnological use.

Enhanced growth performance, oxidative capacity and immune responses of common carp, Cyprinus carpio fed with Artemisia absinthium extract-supplemented diet

In the present study, the effects of Afsanteen (Artemisia absinthium) aqueous extract (AE) were investigated on growth performance, innate immunity, and oxidative status of common carp, Cyprinus carpio. Common carp juveniles were fed experimental diets that contained 0, 0.5, 1 or 1.5% of AE for 60 days (75 fish/treatment). Thereafter, the fish growth performance, feed efficiency, serum immune-related, biochemical, and antioxidant parameters were determined. The results showed that diets containing 0.5 and 1% of AE enhanced the growth performance and feed efficiency of the fish. Fish fed with AE supplemented diets (at all concentrations) also displayed higher levels of serum lysozyme, alternative complement, total immunoglobulin, superoxide dismutase, and total protein levels compared to the control diet; the highest levels were observed at 1% AE level. Serum albumin levels of all AE-supplemented treatments were significantly higher than the control treatment. Dietary AE supplementation significantly increased plasma glutathione peroxidase activities; the highest value was related to the fish fed 0.5% AE. All AE treatments showed significantly lower serum catalase activities compared to the control; the lowest value was related to 1.5% AE. 0.5 and 1% AE significantly decreased serum malondialdehyde levels. All AE- treated fish exhibited significantly lower serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities; the lowest values were observed in 0.5% or 1% AE. In conclusion, dietary inclusions of AE in juvenile common carp increased growth performance and feed efficiency, improved the innate immunity and antioxidant response, and improved hepatic health. AE at 0.5–1% levels is recommended for common carp diet formulation.

Heteroprotein Complex Coacervate Based on β-Conglycinin and Lysozyme: Dynamic Protein Exchange, Thermodynamic Mechanism, and Lysozyme Activity

Heteroprotein complex coacervate (HPCC) is a liquid-like protein concentrate produced by liquid-liquid phase separation. We revealed the protein dynamic exchange and thermodynamic mechanism of β-conglycinin/lysozyme coacervate, and clarified the effect of HPCC on protein structure and activity. β-conglycinin and lysozyme assembled into coacervate at pH 5.75-6.5 and assembled into amorphous precipitates at higher pH. As the pH dropped from 8 to 6, the number of binding sites of the complex decreased in half, and the desolvation degree corresponding to the entropy gain was greatly reduced, conducing to the formation of coacervates rather than precipitates. The coacervates achieved the unique dynamic exchange by exchanging proteins with the diluted phase, making the uniform distribution of proteins in coacervates. The lysozyme activity was completely retained in β-conglycinin/lysozyme coacervates. These results proved that β-conglycinin-based heteroprotein complex coacervate is a feasible method to encapsulate and enrich active proteins in a purely aqueous environment.