Cross-linked Aggregates Research Articles

Low-moisture extruded mung bean protein isolate and wheat gluten: Structure, techno-functional characteristics and establishment of rehydration kinetics of the products

Low-moisture extruded products provide excellent shelf-life and stability, making the exploration of new formulations with various proteins highly valuable. This study prepared and compared products with different ratios of mung bean protein isolate (MPI) to wheat gluten (WG) (10:0, 9:1, 8:2, 7:3, 6:4, and 5:5). Results showed significant improvements (P < 0.05) in L∗-value and product quality with increasing WG ratios, peaking at an MPI/WG ratio of 7:3. Increased WG also enhanced protein cross-linking aggregation, as evidenced by changes in particle size, secondary structure, and fluorescence spectra. However, excessive WG (MPI/WG = 6:4) adversely affected product structure and techno-functional characteristics. Rehydration kinetics were best described by the Peleg model, with rehydrated products showing decreased hardness (291.25 N) and chewiness (227.45 N), but increased tensile resistance (P < 0.05). Eleven quadratic polynomial equations were developed to predict color and techno-functional characteristics based on WG percentage (0–50%). The findings provide a theoretical basis for expanding the application of MPI in new low-moisture extruded products.

Evaluation of the water state and protein characteristics of Tibetan pork under the storage conditions of modified atmosphere packaging: Effect of oxygen concentration

To explore the changes in water status and protein characteristics of Tibetan pork (TP) under modified atmosphere packaging (MAP) with different oxygen concentrations compared to Duroc×Landrace×Yorkshire pork (DLY), the water holding capacity (WHC), water distribution, protein oxidation, and conformation of both types were determined. Results indicate that under MAP, TP pork and DLY pork exhibited higher water retention and lower protein oxidation compared to air packaging. However, with increased oxygen concentration in the MAP, protein oxidation intensified, leading to reduced WHC in the pork. Compared to DLY pork, TP pork in different packaging conditions maintained the integrity of protein secondary and tertiary structures, reducing protein cross-linking aggregation. The lower content of P3 in the two-dimensional relaxation spectra, shorter T1 and T2 relaxation times, and higher proton density suggest better water retention properties in Tibetan pork. These findings support the development of long-distance preservation and transportation technologies for TP pork.

Immobilization of cross-linked enzymes aggregates on hierarchical covalent organic frameworks: Highly stable chemoenzymatic nanoreactor for asymmetric synthesis of optically active halohydrins

Organometallic catalyst is extensively applied for the non-enzymatic regeneration of nicotinamide adenine dinucleotide (phosphate) cofactors, but suffering from the mutual inactivation with the enzymes in one pot. The spatially separated immobilization of organometallic catalyst and enzymes on suitable carriers not only can reduce their mutual inhabitation but also can enhance their reusability. Here in this work, we present a hierarchical porous COFs (HP-TpBpy) that incorporated with [(Cp*RhCl2]2 to generate the metalized COF, Rh-HP-TpBpy. The obtained Rh-HP-TpBpy exhibited superior performance in nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration using formate as the hydride donor, significantly outperforming the natural formate dehydrogenases in cofactor preference toward NADP+. Subsequently, the Lactobacillus fermentum short-chain dehydrogenase/reductase 1 (LfSDR1) was then cross-linked into enzyme aggregates (CLEA) and immobilized on hierarchical Rh-HP-TpBpy, achieving the integrated chemoenzymatic catalyst, LfSDR1@Rh-HP-TpBpy, which can catalyze the chemoenzymatic reduction of halogenated aryl ketones and give the corresponding optically active halohydrins with high conversion and enantiomeric excess (ee) value up to 99 %. The LfSDR1@Rh-HP-TpBpy also exhibits largely enhanced stability compared with the free LfSDR1 and the CLEAs-LfSDR1, enabling its excellent reusability.

Exploration of dynamic interaction between β-lactoglobulin and casein micelles during UHT milk process

The protein aggregation induced by UHT treatment shortens the shelf life of UHT milk. However, the mechanism of β-Lg induced casein micelle aggregation remains unclear. Herein, the dynamic interaction between β-Lg and casein micelles during UHT processing was investigated by experimental techniques and molecular dynamics simulations. Results showed that β-Lg decreased the stability of casein micelles, increased their size and zeta potential. Raman and FTIR spectra analysis suggested that hydrogen and disulfide bonds facilitated their interaction. Cryo-TEM showed that the formation of the casein micelle/β-Lg complex involved rigid binding, flexible linking, and severe cross-linking aggregation during UHT processing. SAXS and MST demonstrated β-Lg bound to κ-casein on micelle surfaces with a dissociation constant (Kd) of 3.84 ± 1.14 μm. Molecular docking and dynamic simulations identified the interacting amino acid residues and clarified that electrostatic and van der Waals forces drove the interaction. UHT treatment increased hydrogen bonds and decreased total binding energy. The non-covalent binding promoted the formation of disulfide bonds between β-Lg and casein micelles under heat treatment. Ultimately, it was concluded that non-covalent interaction and disulfide bonding resulted in casein micelle/β-Lg aggregates. These findings provided scientific insights into protein aggregation in UHT milk.

Ultrasensitive dynamic light scattering immunodetection of alpha-fetoprotein using heptamer-amplified nanoparticle crosslinking aggregation.

Anovel construction strategy is introducedfor an ultrasensitive dynamic light scattering (DLS) immunosensor targeting alpha fetoprotein (AFP). This approach relies on a self-assembled heptamer fusion protein (A1-C4bpα), incorporating the dual functions of multivalent recognition and crosslinking aggregation amplification due to the presence of seven AFP-specific A1 nanobodies on the A1-C4bpα heptamer. Leveraging antibody-functionalized magnetic nanoparticles for target AFP capture and DLS signal output, the proposed heptamer-assisted DLS immunosensor offers high sensitivity, strong specificity, and ease of operation. Under the optimized conditions, the designed DLS immunosensor demonstrates excellent linear detection of AFP in the concentration range 0.06 ng mL-1 to 512 ng mL-1, with a detection limit of 15 pg mL-1. The selectivity, accuracy, precision, practicability, and reliability of this newly developed method were further validated through an assay of AFP levels in spiked and actual human serum samples. Thiswork introduces a novel approach for constructing ultrasensitive DLS immunosensors, easily extendable to the sensitive determination of other targets via simply replacing the nanobody sequence, holding great promise in various applications, particularly in disease diagnosis.

Design and fabrication of magnetic cross-linked laccase aggregate using superparamagnetic metal-organic frameworks for phenol removal

Phenolic pollutants represent a significant environmental concern due to their widespread presence in industrial effluents, agricultural runoff, and urban waste streams. Enzymatic bioremediation emerges as a promising approach for the efficient removal of phenolic pollutants from contaminated environments. Laccases exhibit remarkable catalytic capabilities in degrading phenolic compounds into less harmful or even inert products. In this study, magnetic cross-linked laccase aggregate was prepared using superparamagnetic metal-organic frameworks and then was applied for phenol removal. The obtained results showed that the magnetic cross-linked laccase aggregate retains its activity across a broad pH range. Interestingly, (NH4)2SO4 exhibited a positive influence on enzyme activity, resulting in a relative activity of 137.7 %. The findings suggest that an optimal concentration range of 0.4–0.8 % can achieve maximum enzyme activity while avoiding potential negative impacts associated with excessive cross-linking. Optimal pH and temperature conditions were identified for enhanced phenol removal efficiency as pH 4.5 and 45℃, respectively. The investigation revealed the remarkable reusability of the cross-linked enzyme over multiple cycles. Harnessing the power of enzymes for phenolic pollutant remediation holds immense promise for mitigating environmental pollution and safeguarding public health.

Synthesis and Characterization of Cross-Linked Aggregates of Peroxidase from Megathyrsus maximus (Guinea Grass) and Their Application for Indigo Carmine Decolorization.

We present the synthesis of a cross-linking enzyme aggregate (CLEAS) of a peroxidase from Megathyrsus maximus (Guinea Grass) (GGP). The biocatalyst was produced using 50%v/v ethanol and 0.88%w/v glutaraldehyde for 1 h under stirring. The immobilization yield was 93.74% and the specific activity was 36.75 U mg-1. The biocatalyst surpassed by 61% the free enzyme activity at the optimal pH value (pH 6 for both preparations), becoming this increase in activity almost 10-fold at pH 9. GGP-CLEAS exhibited a higher thermal stability (2-4 folds) and was more stable towards hydrogen peroxide than the free enzyme (2-3 folds). GGP-CLEAS removes over 80% of 0.05 mM indigo carmine at pH 5, in the presence of 0.55 mM H2O2 after 60 min of reaction, a much higher value than when using the free enzyme. The operational stability showed a decrease of enzyme activity (over 60% in 4 cycles), very likely related to suicide inhibition.

Microbial Biofilm Inhibition Using Magnetic Cross-Linked Polyphenol Oxidase Aggregates.

Microbial biofilm accumulation poses a serious threat to the environment, presents significant challenges to different industries, and exhibits a large impact on public health. Since there has not been a conclusive answer found despite various efforts, the potential green and economical methods are being focused on, particularly the innovative approaches that employ biochemical agents. In the present study, we propose a bio-nanotechnological method using magnetic cross-linked polyphenol oxidase aggregates (PPO m-CLEA) for inhibition of microbial biofilm including multidrug resistant bacteria. Free PPO solution showed only 55-60% biofilm inhibition, whereas m-CLEA showed 70-75% inhibition, as confirmed through microscopic techniques. The carbohydrate and protein contents in biofilm extracellular polymeric substances (EPSs) were reduced significantly. The m-CLEA demonstrated reusability up to 5 cycles with consistent efficiency in biofilm inhibition. Computational work was also done where molecular docking of PPO with microbial proteins associated with biofilm formation was conducted, resulting in favorable binding scores and inter-residual interactions. Overall, both in vitro and in silico results suggest that PPO interferes with microbial cell attachment and EPS formation, thereby preventing biofilm colonization.

Endogenous reactive oxygen species (ROS)-driven protein oxidation regulates emulsifying and foaming properties of liquid egg white

Highly oxidative reactive oxygen species (ROS) attack protein structure and regulate its functional properties. The molecular structures and functional characteristics of egg white (EW) protein (EWP) during 28 d of aerobic or anaerobic storage were explored to investigate the “self-driven” oxidation mechanism of liquid EW mediated by endogenous ROS signaling. Results revealed a significant increase in turbidity during the storage process, accompanied by protein crosslinking aggregation. The ROS yield initially increased and then decreased, leading to a substantial increase in carbonyl groups and tyrosine content. The free sulfhydryl groups and molecular flexibility in EWP exhibited synchronicity with ROS production, reflecting the self-repairing ability of cysteine residues in EWP. Fourier-transform infrared spectroscopy indicated stable crosslinking between EWP molecules in the early oxidation stage. However, continuous ROS attacks accelerated EWP degradation. Compared with the control group, the aerobic-stimulated EWP showed a significant decrease in foaming capacity from 30.5 % to 9.6 %, whereas the anaerobic-stimulated EWP maintained normal levels. The emulsification performance exhibited an increasing-then-decreasing trend. In conclusion, ROS acted as the predominant factor causing deterioration of liquid EW, triggering moderate oxidation that enhanced the superior foaming and emulsifying properties of EWP, and excessive oxidation diminished the functional characteristics by affecting the molecular structure.

Impact of Flammulina velutipes polysaccharide on properties and structural changes of pork myofibrillar protein during the gel process in the absence or presence of oxidation

The present study aimed to investigate the impact of Flammulina velutipes polysaccharide (FVSP) on the rheological properties and structural alterations of myofibrillar protein (MP) and oxidized MP (OMP), utilizing techniques such as rhehometer, fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the unoxidized system, the addition of 5.00% FVSP significantly improved (p < 0.05) the storage and loss moduli of the composite gel and promoted the α-helix to β-sheet transformation. These effects enhanced the protein's gel strength and water-holding capacity (WHC). In the oxidation system, 5.00% FVSP had significant effects (p < 0.05) on repair and improvement of the oxidized MP. These effects inhibited the cross-linking aggregation and degradation of the protein. In addition, the addition of FVSP significantly improved the gel properties of MPs after oxidation (p < 0.05), hindered fracture of the protein gel network structure. In summary, polysaccharides have a substantial effect on the functional characteristics of MP, and FVSP could potentially be applied in meat products.

Redox-reversible siderophore-based catalyst anchoring within cross-linked artificial metalloenzyme aggregates enables enantioselectivity switching.

The immobilisation of artificial metalloenzymes (ArMs) holds promise for the implementation of new biocatalytic reactions. We present the synthesis of cross-linked artificial metalloenzyme aggregates (CLArMAs) with excellent recyclability, as an alternative to carrier-based immobilisation strategies. Furthermore, iron-siderophore supramolecular anchoring facilitates redox-triggered cofactor release, enabling CLArMAs to be recharged with alternative cofactors for diverse selectivity.

Characterization of Maltogenic Amylase Activity Recovery: A Potential Approach for Improving Immobilization

Cross-linked enzymes aggregate (CLEA) is a versatile carrier free-immobilization technique that has gained much attention in the development of biocatalyst technology. However, there is no precise and accurate method of using this technique that will lead to an expected outcome that meets the requirements of the industrial standard. Therefore, the objective of this study is to investigate the effect of a few methods of executing cross-linked enzyme aggregates approach using maltogenic amylase by measuring the activity recovery of the developed CLEA. Some factors that are considered in developing the methodologies are the interaction between cross-linkers and enzymes, size of the cross-linked enzyme aggregates and substrate diffusion. The addition of precipitant and the cross-linking agent steps has been manipulated and four different methodologies were developed. Based on the results, Method 2 showed the highest activity recovery (57.9%) whilst Method 4 gave the lowest activity recovery (15.7%). Method 2 is an improvised method that removed supernatant after centrifugation before proceeding to the cross-linking step. The characterization of cross-linked enzyme aggregates such as morphological characterization and Fourier Transform-Infrared Spectroscopy was also determined. In conclusion, the best and most productive preparation method was determined based on the highest activity recovery.

Cross-linked α-amylase aggregates on Fe3O4 magnetic nanoparticles modified with polydopamine/polyethyleneimine for efficient hydrolysis of starch

In this work α-amylase was immobilized on magnetic Fe3O4 nanoparticles with polyethylenimine (PEI)/polydopamine (PDA) coating or 3-aminopropyl triethoxysilane (APTES) for the first time via adsorption–precipitation–cross-linking. Compared with the free α-amylase, the resultant magnetic cross-linked α-amylase aggregates (PEI/PDA-M-CLEAs and N-M-CLEAs) exhibited excellent thermal and storage stability as well as pH stability. After storage at 25 °C for 60 days, free α-amylase only retained 60% of its initial activity, while PEI/PDA-M-CLEAs and N-M-CLEAs retained 80% and 78% of their initial activities, respectively. Furthermore, N-M-CLEAs and PEI/PDA-M-CLEAs showed good reusability. After 6 repeated uses, PEI/PDA-M-CLEAs and N-M-CLEAs still maintained 65% and 62% of their initial activities, respectively. Especially, PEI/PDA-M-CLEAs and N-M-CLEAs exhibited higher starch hydrolysis efficiency than free α-amylase. The maximum dextrose equivalent (DE) values of starch hydrolysis by PEI/PDA-M-CLEAs and N-M-CLEAs reached 29.24% and 28.79% within 90 min, respectively. However, the maximum DE values of starch hydrolysis by the free α-amylase was only 27.89% even in 150 min. The magnetic cross-linked α-amylase aggregates could be introduced as effective biocatalyst for industrial applications in production of maltose syrups.

Cross-linked arginine deiminase aggregates for enhanced production of nutraceutical citrulline

Arginine deiminase (ADI) has been widely employed for the biological production of nutraceutical citrulline. However, sub-optimal enzyme properties, operational instability, and difficulty in reuse impede its industrial usage as well as increase the production cost. The present study describes a simple, cost-effective carrier-free immobilization technology that can circumvent these limitations. ADI was precipitated and cross-linked with glutaraldehyde to obtain novel cross-linked arginine deiminase aggregates (ADI-CLEA). A neural network-based multiobjective genetic algorithm (MOGA-NN) was employed to optimize the immobilization of ADI. MOGA-NN optimization yielded ADI-CLEA with 0.30 IU/mL activity and 61 % recovery. Structural characterization using TEM, SEM, FTIR, DLS/ZETA revealed that the developed ADI-CLEA were monodisperse, ultra-porous, and highly stable. ADI-CLEA showed an improved pH /temperature profile and resistivity against denaturants than free enzyme. The efficiency of ADI-CLEA in the industrial production of nutraceutical citrulline was also studied. ADI-CLEA produced 215.4 g/L citrulline in first cycle of usage. Reusability and storage stability analysis further showed that ADI-CLEA could be reused for more than seven consecutive cycles and stable for up to 50 days. In conclusion, this novel formulation increases the stability/robustness of ADI, eases the product separation process and could be reused for economically viable bioproduction of nutraceutical citrulline in industries.

Innovative biocatalyst synthesis of pectinolytic enzymes by cross-linking strategy: Potentially immobilised pectinases for the production of pectic-oligosaccharides from pectin

Pectinases are outstanding multienzymes, which have the potential to produce new emerging pectic-oligosaccharides (POS) via enzymatic hydrolysis of pectin. However, free pectinase is unable to undergo repeated reaction for the production of POS. This study proposed a sustainable biocatalyst of pectinases known as cross-linked pectinase aggregates (CLPA). Pectinase from Aspergillus aculeatus was successfully precipitated using 2 mg/mL pectinase and 60 % acetone for 20 min at 20 °C, which remained 36.3 % of its initial activity. The prepared CLPA showed the highest activity recovery (85.0 %), under the optimised conditions (0.3 % (v/v) starch and glutaraldehyde mixture (St/Ga), 1.5: 1 of St/Ga, 25 °C, 1.5 h). Furthermore, pectin-degrading enzymes from various sources were used to produce different CLPA. The alteration of pectinase secondary structure gave high stability in acidic condition (pH 4), thermostability, deactivation energy and half-life, and improved storage stability at 4 °C for 30 days. Similarly to their free counterpart, the CLPA exhibited comparable enzymatic reaction kinetics and could be reused eight times with approximately 20 % of its initial activity. The developed CLPA does not only efficaciously produced POS from pectin as their free form, but also exhibited better operational stability and reusability, making it more suitable for POS production.

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.

Effects of ferulic acid on the polymerization behavior of gluten protein and its components

The effects of ferulic acid (FA) at different concentrations on the aggregation behaviors of gluten and its components (gliadin and glutenin) were determined in this study. Results showed that the free sulfhydryl content of glutenin and gluten significantly reduced while the mean particle size of gluten increased following FA supplementation (0.5%). Moreover, β-sheet content of gluten increased by 20.40% compared with the control group when 0.5% FA was added. Contrarily, higher concentrations (1.0 and 1.5%) of FA significantly reduced tyrosine (TYR) bimodal ratio of glutenin. Furthermore, the concentrations increased the polarity of tryptophan (TRP) residues microenvironment, which promoted the formation of a disordered gluten structure. Additionally, SDS-PAGE showed that 0.5% FA supplementation promoted the formation of high molecular weight gluten by cross-linking and aggregation of low molecular weight gluten components. Using atomic force microscopy, it was confirmed that at 0.5% FA promoted the cross-linking aggregation of gluten molecules. Further, the microscopy analysis showed that higher doses of FA contributed to an uneven distribution of gluten aggregates and a disordered and loose structure of gluten network, which was not favorable for the formation of continuous and stable dough.

Characterization of 36-kDa Dimeric Methotrexate-degrading Enzyme from Variovorax paradoxus, its Performance and Stability of its Cross-linked Aggregates

Characterization of 36-kDa Dimeric Methotrexate-degrading Enzyme from Variovorax paradoxus, its Performance and Stability of its Cross-linked Aggregates

Cross-linked phytase aggregates for improved phytate degradation at low pH in animal feed

Phytases are widely used food and feed enzymes to improve phosphate availability and reduce anti-nutritional factors. Despite the benefits, enzyme usage is restricted by the harsh conditions in a gastrointestinal tract (pH 2–6) and feed pelleting conditions at high temperatures (60–90 °C). The commercially available phytase Quantum® Blue has been immobilized as CLEAs using glutardialdehyde and soy protein resulting in a residual activity of 33%. The influence of the precipitating agent, precipitant concentration, cross-linker concentration and cross-linking time, sodium borohydride as well as the proteic feeders gluten, soy protein and bovine serum albumin (BSA) has been optimized. The best conditions were 90% (v/v) ethyl lactate as precipitating reagent, 100 mM glutardialdehyde and a soy protein concentration of 227 mg/L with a cross-linking time of 1 h. The intrinsically stable phytase remained its high thermal stability and temperature optimum. The phytase-CLEA achieved a 425% increase of residual activity under harsh acidic conditions between pH 2.2 and 3.5 compared to the free enzyme. The free and immobilized phytase were deployed in an in vitro assay simulating the acidic conditions in the gizzard of poultry at pH 2. The hydrolysis of phytate was monitored using a novel high-performance thin-layer chromatography (HPTLC) analysis and DAD scanner to study the InsPx fingerprint. All lower inositol phosphate pools (InsP1–InsP6) and free phosphate were separated and analyzed. The phytase-CLEA efficiently released 80% of the total phosphate within 180 min, whereas the free enzyme only released 6% in the same time under the same conditions.

Removal of pentachlorophenol and phenanthrene from lignocellulosic biorefinery wastewater by a biocatalytic/biosurfactant system comprising cross-linked laccase aggregates and rhamnolipid

Synthesis and characterization of highly active cross-linked laccase aggregates (CLLAs) were performed and evaluated for removal of pentachlorophenol and phenanthrene from lignocellulosic biorefinery wastewater. Laccase from Tramates versicolor MTCC 138 was insolubilized as CLLAs via precipitation with 70% ammonium sulphate and simultaneous cross-linking with 5 mM glutaraldehyde to obtain activity recovery of 89.1%. Compared to the free laccase, the pH and thermal stability of the prepared CLLAs were significantly higher. At a high temperature of 60 °C, free laccase had a half-life of 0.25 h, while CLLAs had a half-life of 6.2 h. In biorefinery wastewater (pH 7.0), the free and CLLAs were stored for 3 day at a temperature of 30 °C. Free laccase completely lost their initial activity after 60 h; however, the CLLAs retained 39% activity till 72 h. Due to its excellent stability, free laccase and CLLAs were assessed for removing pentachlorophenol and phenanthrene in wastewater. CLLAs could remove 51–58% of pentachlorophenol (PCP) and phenanthrene (PHE) in 24 h. Biosurfactants, including surfactin, sophorolipid, and rhamnolipid, were assessed for their aptitude to improve the removal of organic contaminants in wastewater. Biorefinery wastewater incubated with all surfactants enhanced PCP and PHE removal compared to the no-surfactant controls. Further, 1 μM rhamnolipid significantly amplified pentachlorophenol and phenanthrene removal to 81–93% for free laccase and CLLAs, respectively.