Intermolecular Cross-linking Research Articles

Oxidative modification of malondialdehyde influences the structure and emulsification properties of egg yolk high-density lipoprotein

Malondialdehyde (MDA) represents secondary active oxidation products in lipid peroxidation. This research used MDA to establish a simulated oxidation system to study its effects on egg yolk high-density lipoprotein (EYHDL) structure and emulsifying properties. It was shown that incubation of EYHDL with MDA resulted in protein carbonylation and a gradual reduction in the sulfhydryl content. Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy indicated that MDA oxidation would destroy the ordered structure of EYHDL and the structural stability of the protein. Oxidation might result in the intermolecular cross-linking of EYHDL and the formation of oxidized aggregates, leading to a reduction in surface hydrophobicity. For the EYHDL emulsion modified by MDA oxidation, it could be seen that the emulsion stratification was evident at the macroscopic level, and the grain diameter of the EYHDL emulsion was observed to increase with the MDA concentration by inverted fluorescence microscopy. The emulsification activity (EAI) and emulsification stability (ESI) of EYHDL were decreased by malondialdehyde oxidation modification, which may be due to the decrease of protein surface hydrophobicity and oxidation aggregation.

Characterization of enzymatic cross-linking soy protein isolate xerogels and its shape memory effect induced by ethylcellulose

In this study, the deformation behavior of soybean protein isolate (SPI) xerogels cross-linked with different transglutaminase (TGase) concentrations was investigated. The gel properties of TGase cross-linked SPI were analyzed by rheology and texture. Results showed that 0.4% TGase completely promoted the intermolecular cross-linking, SPI molecules had more binding sites and α-helix content and less irregular curl structure. The presence of 0.4% TGase enhanced the water binding ability and thermal stability of SPI xerogel, and its denaturation temperature was up to 181.50 °C. The corresponding texture characteristics showed that hardness and elasticity were significantly increased by 182.90% and 25.00%, respectively. Results showed that SPI containing 0.4% TGase had the best 3D (three-dimension) shape change after hydration. However, excessive TGase (1.0% w/v) led to excessive lysine covalent cross-linking, which increases the porosity of the gel surface, causing the disrupted gel network. The research provides insights and new ideas for food-processing technology called 4D (four-dimension) food.

Hydrogen-Induced Defective Crystalline Carbon Nitride with Enhanced Bidirectional Charge Migration for Persulfate Photoactivation

A feasible way to overcome the moderate photoactivity of carbon nitride is by decreasing the carrier diffusion distance and increasing the intralayer active sites by synthesizing defective crystalline carbon nitride. However, the additive-free synthesis of this type of structure remains a great challenge, due to the trade-off between crystallinity control and defect modulation. Herein, we demonstrate the hydrogen-assisted synthesis of ultrathin carbon nitride nanosheets with efficient exciton dissociation and fast electron transfer. Experimental and theoretical investigations indicate that hydrogen not only facilitates intermolecular cross-linking for crystallization but also generates abundant nitrogen vacancies among non-hydrogen-bonded tri-s-triazine units for charge transportation. The simultaneous modulation of interlayer and intralayer interactions contributes to the efficient bidirectional charge transfer during photocatalytic reactions. The synergy of defective crystalline carbon nitride and persulfate oxidants results in a striking performance for contaminant degradation, with 9.2- and 18.4-times higher activity than separate photocatalysis and persulfate photoactivated by bulk g-C3N4, respectively. This work not only provides a facile approach to synthesizing defective crystalline carbon nitride without employing any extrinsic additives but also opens up a new avenue to improve the efficiency of metal-free photocatalysts by bidirectional charge modulation.

Intermolecular cross-linked polymer of intrinsic microporosity-1 (PIM-1)-based thin-film composite hollow fiber membrane for organic solvent nanofiltration

Organic solvent nanofiltration (OSN) is a membrane-based organic solution separation process that has emerged as a viable alternative to energy- and solvent-intensive separation processes. Polymers of intrinsic microporosity (PIMs) are the targets of recent active research on OSN membrane materials because of their unique physicochemical properties, such as solution processability, chemical tunability, robust porosity, and high thermal stability. However, applying PIMs-based membranes in practical OSN applications is still challenging because of their limited organic solvent stability and inevitable decrease in performance. Meanwhile, despite the potential of thin-film composite hollow fiber membranes for OSN, only a few studies have been reported to date. In this study, we fabricated a PIMs-based thin-film composite hollow fiber membrane by dip-coating and improved its organic solvent stability by intermolecular cross-linking. The membrane exhibited pure ethanol permeance of 10.9 LMH/bar and a molecular weight cut-off of 952 g/mol in ethanol. The membrane displayed stable dye rejection performance (>95%) even after immersion in ethanol, acetone, and toluene for seven days. This study demonstrates that intermolecular cross-linking between PIMs polymer chains can improve the organic solvent stability of PIMs-based membranes and provides a desirable strategy to fabricate feasible organic solvent stable PIMs-based thin-film composite hollow fiber membranes for OSN applications.

Viewing biofilm formation through a multifocal lens of physics and biology

Recent studies on the formation, organisation and dynamics of biofilms highlight the interplay between physical forces and biological programs. Two complementary generalised pathways that explain the mechanisms driving biofilm formation have emerged. In the first pathway, where physical forces precede the biological program, the initial expansion of cells leads to cell clustering or aggregation prior to the production of extracellular polymeric substances (EPS). The second pathway describes an initial biologically prompted production of EPS, which introduces new biophysical interactions within the EPS, such as by phase separation, macromolecular crowding, excluded volume interactions and intermolecular cross-linking. In practice, which of the two pathways is adopted is ultimately determined by the specificities of the biofilm and the local microenvironment, each leading to the formation of robust, viscoelastic biofilm. Within this framework, we further highlight here recent findings on the role of higher-order structures in matrix gelation and phase separation of EPS in promoting the clustering of bacteria. We assert that examining biofilms through the combined lens of physics and biology promises new and significant methodological and conceptual advancements in our understanding of biofilms.

Modulation of the biophysical and biochemical properties of collagen by glycation for tissue engineering applications.

The structural and functional properties of collagen are modulated by the presence of intramolecular and intermolecular crosslinks. Advanced Glycation End-products (AGEs) can produce intermolecular crosslinks by bonding the free amino groups of neighbouring proteins. In this research, the following hypothesis is explored: The accumulation of AGEs in collagen decreases its proteolytic degradation rates while increasing its stiffness. Fluorescence Lifetime Imaging (FLIM) and Fourier-transform infrared spectroscopy (FTIR) detect biochemical changes in collagen scaffolds during the glycation process. The accumulation of AGEs increases exponentially in the collagen scaffolds as a function of Methylglyoxal (MGO) concentration by performing autofluorescence measurement and competitive ELISA. Glycated scaffolds absorb water at a much higher rate confirming the direct affinity between AGEs and interstitial water within collagen fibrils. In addition, the topology of collagen fibrils as observed by Atomic Force Microscopy (AFM) is a lot more defined following glycation. The elastic modulus of collagen fibrils decreases as a function of glycation, whereas the elastic modulus of collagen scaffolds increases. Finally, the enzymatic degradation of collagen by bacterial collagenase shows a sigmoidal pattern with a much slower degradation rate in the glycated scaffolds. This study identifies unique variations in the properties of collagen following the accumulation of AGEs. STATEMENT OF SIGNIFICANCE: In humans, Advanced Glycation End-products (AGEs) are naturally produced as a result of aging process. There is an evident lack of knowledge in the basic science literature explaining the biomechanical impact of AGE-mediated crosslinks on the functional and structural properties of collagen at both the nanoscale (single fibrils) and mesoscale (bundles of fibrils). This research, demonstrates how it is possible to harness this natural phenomenon in vitro to enhance the properties of engineered collagen fibrils and scaffolds. This study identifies unique variations in the properties of collagen at nanoscale and mesoscale following accumulation of AGEs. In their approach, they investigate the unique properties conferred to collagen, namely enhanced water sorption, differential elastic modulus, and finally sigmoidal proteolytic degradation behavior.

Effect of ultrasonic pretreatment on the rheology and structure of grass pea (Lathyrus sativus L.) protein emulsion gels induced by transglutaminase.

In this study, emulsion gels were prepared by sonicated grass pea protein isolates (GPPI) at different ultrasonic amplitudes (25, 50 and 75%) and times (5, 10 and 20min). Formation of emulsion gels was induced by transglutaminase. Enzymatic gelation of emulsions stabilized by sonicated GPPI occurred in two stages. A relatively fast stage led to the formation of a weak gel which was followed by a slow stage that strongly reinforced the gel structure. Emulsion gels fabricated by sonicated GPPIs showed a homogeneous and uniform droplet distribution with higher elastic modulus compared to the native protein. A stiffer emulsion gel with a higher G' was formed after the protein was treated at 75% amplitude for 10min. After sonication of GPPI, the water holding capacity (WHC) of emulsion gels increased in accordance with the mechanical properties. Higher intermolecular cross-linking within the gel network increased the thermal stability of emulsion gels fabricated by sonicated GPPI. Although sonicated-GPPI emulsion gels clearly displayed homogenous microstructure in comparison to that made with native GPPI, the microstructures of these gels were nearly identical for all sonication amplitudes and times.

Refilling strategy of crosslinked aromatic SAMs for enhancing the molecular packing density

The stability and uniform coverage of molecular self-assembled monolayers (SAMs) on metal substrates is of crucial importance for device integration. One strategy to enhance the stability of the SAMs is based on electron irradiation-induced intermolecular crosslinking. During this process, the coverage area of the SAMs decreases due to the formation of carbon–carbon covalent bonds between the neighboring molecules, which leaves empty spaces at the interfaces. Therefore, to increase molecular coverage, the empty spaces can be refilled either by the same or different types of molecules. In this article, we demonstrate a multistep method of creating densely packed SAMs of biphenyl-4-4-thiol molecules after electron radiation. 4′-Mercapto-[1,1′-biphenyl]-4-carbonitrile molecules are used for refilling because of the distinct signature of nitrogen atoms as a marker in the X-ray photoelectron spectroscopy (XPS) spectra. We used these molecules to estimate the reduction of the area of SAM coverage after electron radiation. As a result, 28.9% ± 5.9% of the total surface area was released. The experimental results are supplemented by density functional theory calculations to estimate the area of the empty regions caused by intermolecular crosslinking. The theoretical predictions align closely with the experimental results. These findings can be of practical importance in creating stable molecular SAM devices using the proposed alternating irradiation and refilling procedure.

Insights on Chemical Crosslinking Strategies for Proteins

Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the host system. Many materials that are used in biomedical and food packaging industries are prepared by chemical means of crosslinking the proteins, besides the physical or enzymatic means of crosslinking. Such chemical methods utilize the chemical compounds or crosslinkers available from natural sources or synthetically generated with the ability to form covalent/non-covalent bonds with proteins. Such linkages are possible with chemicals like carbodiimides/epoxides, while photo-induced novel chemical crosslinkers are also available. In this review, we have discussed different protein crosslinking strategies under chemical methods, along with the corresponding crosslinking reactions/conditions, material properties and significant applications.

Full Recycling and Re-Use of Bio-Based Epoxy Thermosets: Chemical and Thermomechanical Characterization of the Recycled Matrices.

High performances of thermosets deriving from their covalent intermolecular cross-link bonds result in their low recyclability hindering the full exploitation of a truly circular approach for cured thermosets. In this experimental work, the recyclability of a bio-based fully recyclable epoxy resin using a mild chemical recycling process was demonstrated. The recycled polymer obtained was fully characterized to ascertain its structure and properties. MALDI (Matrix-Assisted Laser Desorption/Ionization), GPC (Gel Permeation Chromatography) and NMR (Nuclear Magnetic Resonance) spectroscopy to determine the chemical structure of the recycled polymer were used. The thermomechanical properties of the cured virgin network and of the recycled product obtained were measured by DSC (Differential Scanning Calorimetry) and DMA (Dynamic Mechanical Analysis). Thermogravimetric analysis of the recycled polymer was also performed. The recycled polymer was transformed into a polyurethane by reacting it with an isocyanate. The synthetized polyurethane obtained therefrom was thoroughly characterized by thermogravimetric analysis. This approach proved the possibility to up-scale the recycled product making it available for novel applications exploiting its re-use.

Mechanical and rheological effects of transglutaminase treatment on dense plant protein blends

Pea protein isolate (PPI) and mung bean protein isolate (MBPI) are rising alternatives for soy protein isolate in producing meat analogues. Transforming these isolates into full products is challenging due to their differences in functional properties compared with soy. Here, we report on the use of transglutaminase to improve the mechanical and rheological properties of dense MBPI and PPI dispersions. Gels from PPI and MBPI (40 wt% dry matter) were produced with 0, 0.1, 0.3, 0.5, and 0.7 wt% transglutaminase and incubation temperatures of 30, 40, 50, and 60 °C. Their mechanical and rheological properties were determined by tensile and closed cavity rheometer tests. The degree of crosslinking was determined by the OPA assay. Transglutaminase significantly increased fracture stress and strain of PPI gels, and to a lesser extent those of MBPI gels. The degree of free amino confirmed that this increase in PPI was due to formation of additional crosslinks. Amplitude sweeps identified that the rheological properties of PPI gels and MBPI gels were affected by the addition of transglutaminase, but in an opposite manner. Stress relaxation experiments showed that transglutaminase increased the elasticity of PPI gels and formed mainly small aggregates in MBPI gels. This work showed that the fibrous structure diminished when >0.1 wt% transglutaminase was added to PPI/MBPI - wheat gluten blends. To conclude, transglutaminase can be used to create intermolecular crosslinks in PPI gels, creating a full network, while mostly intramolecular crosslinks are formed in MBPI gels, leading to a cluster structure.

Reproducible and controlled peptide functionalization of polymeric nanoparticles

Polymeric nanoparticles containing multiple amines and carboxylates have been frequently used in drug delivery research. Reproducible and controlled conjugation among these multifunctional biomaterials is necessary to achieve efficient drug delivery platforms. However, multiple functional groups increase the risk of unintended intramolecular/intermolecular reactions during conjugation. Herein, conjugation approaches and possible undesired reactions between multi-amine functionalized peptides, multi-carboxylate functionalized polymers, and anhydride-containing polymers [Poly(styrene-alt-maleic anhydride)-b-poly(styrene)] were investigated under different conjugation strategies (carbodiimide chemistry, anhydride ring-opening via nucleophilic addition elimination). Muti-amine peptides led to extensive crosslinking between polymers regardless of the conjugation chemistry. Results also indicate that conventional peptide quantification methods (i.e., o-phthalaldehyde assay, bicinchoninic acid assay) are unreliable. Gel permeation chromatography (GPC) provided more accurate qualitative and quantitative evidence for intermolecular crosslinking. Crosslinking densities were correlated with higher feed ratios of multifunctional peptides and carbodiimide coupling reagents. Selectively protected peptides (Lys-Alloc) exhibited no crosslinking and yielded peptide-polymer conjugates with controlled dispersity and molecular weight. Furthermore, anhydride ring-opening (ARO) nucleophilic addition elimination was successfully introduced as a facile yet robust peptide conjugation approach for cyclic anhydride-containing polymers.

Fluoroalkylated-GAP copolymers (GAP-FP) as promising energetic binders

The development of a novel energetic block copolymer of glycidyl azide polymer (GAP) and fluoropolymer (FP) using a boron trifluoride-tetrahydrofuran complex/diol initiator system is reported herein. Well-defined compositions of the GAP-FP copolymers in two different GAP to FP ratios (1:1 and 1:3) were synthesized by tailoring the desired molecular weights of each block in the copolymer, demonstrating the synthetic versatility of such a copolymer system. The resultant GAP-FP copolymers represent a unique hybrid binder system with tunable energy releasing and oxidizing potentials intended for metallized formulations. Thermogravimetric analysis showed that the carbonaceous residue usually formed from the decomposition of GAP could be reduced significantly by the copolymerization with FP. Isoconversional method of kinetic analysis of the GAP-FP copolymers revealed an increasing dependence of the effective activation energy on the extent of conversion. The increasing dependence suggested a mechanism of the competing reactions that were found to be between the reactions of FP triggered oxidation and intermolecular crosslinking of the polyimine intermediates formed from GAP decomposition that ultimately resulted in the reduction of the carbonaceous residue.

Photocrosslinking of Polyacrylamides Using [2 + 2] Photodimerisation of Monothiomaleimides.

The [2 + 2] photocycloaddition of monothiomaleimides (MTMs) has been exploited for the photocrosslinking of polyacrylamides. Polymer scaffolds composed of dimethylacrylamide and varying amounts of d,l-homocysteine thiolactone acrylamide (5, 10, and 20 mol %) were synthesized via free-radical polymerization, whereby the latent thiol functionality was exploited to incorporate MTM motifs. Subsequent exposure to UV light (λ = 365 nm, 15 mW cm–2) triggered intermolecular crosslinking via the photodimerization of MTM side chains, thus resulting in the formation of polyacrylamide gels. The polymer scaffolds were characterized using Fourier transform infrared spectroscopy, UV–visible spectroscopy, 1H NMR spectroscopy, and size exclusion chromatography, confirming the occurrence of the [2 + 2] photocycloaddition between the MTM moieties. The mechanical and physical properties of the resulting gels containing various MTM mol % were evaluated by rheology, compression testing, and swelling experiments. In addition, scanning electron microscopy was used to characterize the xerogel morphology of 5 and 10 mol % MTM hydro- and organo-gels. The macro-porous morphology obtained for the hydrogels was attributed to phase separation due to the difference in solubility of the PDMA modified with thiolactone side chains, provided that a more homogeneous morphology was obtained when the photo-gels were prepared in DMF as the solvent.

Impact of divalent cations on lysozyme-induced solubilisation of waste-activated sludge: Perspectives of extracellular polymeric substances and surface electronegativity

Lysozyme hydrolysis can accelerate waste-activated sludge (WAS) solubilisation, which can significantly shorten the process and promote the efficiency of anaerobic digestion. This study investigated the impact of divalent cations on lysozyme-induced solubilisation of WAS. The performance of lysozyme pretreatment was dramatically inhibited by Mg2+ and Ca2+. Compared to the control group, the amount of net SCOD, protein, and polysaccharides released to the supernatant were reduced by 36.6%, 44.7%, and 35.8%, respectively, in the presence of divalent cations. The extracellular polymeric substance (EPS) matrix became tightly bound, resulting in fewer proteins and polysaccharides being extracted from loosely-bound EPS (LB-EPS) with divalent cations, which was detrimental to the solubilisation of WAS. Divalent cations decreased the surface electronegativity of sludge particles and prolonged the adsorption of lysozymes by sludge flocs. More than 16.6% of total lysozymes remained in the liquid phase of WAS after 240min Mg2+ and Ca2+ strengthened the binding among proteins and polysaccharides and promoted the intermolecular cross-linking of polysaccharides. The EPS matrix formed a dense spatial reticular structure that blocked the transfer of lysozymes from the EPS matrix to the pellet. As a result, the lysozymes accumulated in LB-EPS rather than hydrolysing the microorganism's cell wall. This study provides a new perspective on the restriction of WAS pretreatment with lysozymes and optimises the method of lysozyme-induced solubilisation of WAS.

Double-Network Luminescent Films Constructed Using Sulfur Quantum Dots and Lanthanide Complexes.

Although UV light-switchable luminescent films are of importance for application in soft optical devices and anticounterfeiting labels, there are still challenges in developing such films integrated with outstanding luminescent property, high self-healing efficiency, and simultaneously excellent mechanical strength. Herein, double-network (DN) luminescent films are designed and constructed via an intermolecular hydrogen bond crosslinking strategy of poly(ethylene glycol) (PEG) in sulfur quantum dots (S-QDs) and polyurethane (PU), where S-QDs ("stone" one) play dual roles of acting both as a soft segment to crosslink another segment PU ("bird" one) and also as the origin of a luminescence center ("bird" two) in films. In addition, lanthanide(III) complexes (LnCs, Ln═Eu3+, Tb3+) are employed as another emission source to embed in the films and switch the emission colors of DN films from the multicolor (red-yellow-green) of LnCs to the blue color of S-QDs by changing the ultraviolet excitation wavelength from 254 to 365 nm. It is worth noting that the crosslinking network strategy can effectively prevent S-QDs and LnCs from aggregating or leaking and enable both luminescence centers to homogeneously distribute, resulting in luminescent DN films possessing extraordinary UV light-switchable luminescence, improved mechanical property, and excellent self-healing ability. This work presents a viable method for the design and fabrication of luminescent films with multifunctional applications in flexible robotics, wearable devices, and dual-luminescent anticounterfeiting materials.

Active-intelligent and biodegradable sodium alginate films loaded with Clitoria ternatea anthocyanin-rich extract to preserve and monitor food freshness

The aim of this study was to develop and characterize sodium alginate films loaded with 10–40 % Clitoria ternatea extract (CTE) and apply to monitoring the quality of milk, pork and shrimp. Films loaded with CTE showed high light barrier capacity and improved tensile strength by 3.8 times over control films. The incorporation of CTE in alginate films improved the thermal stability of the materials due to intermolecular interactions and crosslinking of polymeric networks. The addition of 40 % of CTE generated films with antibacterial action against E. coli. The alginate films showed biodegradable characteristics in soil and beach sand in 15 days. The food simulant test revealed that the loaded films show good compatibility with aqueous and acidic foods due to the release of higher levels of polyphenols and anthocyanins. The films showed great colorimetric potential due to their ability to change color at different pH (pink-green), ammonia gas (blue-green) and sterilization process (blue-yellow). When the film loaded with 40 % CTE (F40) was applied to monitor the freshness of milk and meat products (shrimp and pork), its blue color changed to purple and green, respectively. Therefore, the F40 has great potential to be used as a biodegradable indicator of freshness.

Hybrid polymer networks of carbene and thiol ene

Thiol/ene-based resorbable elastomers display tough elongation but lack adhesion to soft tissues. Carbene-based bioadhesives (e.g. CaproGlu) allow soft tissue adhesion, but the covalent crosslinks limit extensibility after photoactivation. Herein thiol/ene resorbable elastomers are combined with a carbene bioadhesive into a 3-component hybrid network by exploiting tunable photoactivation of each macromolecule independently or simultaneously. Dual crosslinking was monitored by photorheometry, where 405 nm initiates formation of a thiol/ene elastomeric network, followed by 365 nm activation of diazirine-grafted polycaprolactone tetrol (CaproGlu). Dynamic shear moduli, gelation point, elongation at break, and lap shear stress of the hybrid polymer network are evaluated with respect to absorbed light energy dose. Surface-exposed unreacted CaproGlu enables adhesion of the hybrid network to various substrates, as well as intermolecular crosslinking within the transparent matrix. The network morphology and functional group conversion is evaluated through scanning electron microscopy and infrared spectroscopy, respectively. For the first time, we demonstrate hybrid thiol/ene/diazirine double sided bioadhesives with tunable dynamic moduli in the range of 10–800 kPa and 160 kPa lap-shear adhesion strength.

Hydrothermal-induced changes in the gel properties of Mung bean proteins and their effect on the cooking quality of developed compound noodles.

Mung bean proteins (MBPs) are highly nutritious food ingredients, but their lack of gluten limits their use in staple foods such as noodles. In this study, MBPs were modified by hydrothermal treatment, and their gel properties and the major structural changes were analyzed at different heating temperatures (25, 65, 75, 85, 95, and 105°C), moisture contents (0, 15, 20, 25, 30, and 35%), and hydrothermal treatment times (0, 15, 30, 45, 60, and 75 min). Thereafter, the modified MBPs (MMBPs) were added to wheat noodles at substitution levels of 3, 6, and 9% to evaluate their effect on the quality of the noodles. The results showed that the hydrothermal treatment significantly improved the gel properties and water absorption capacity of the MBPs and slightly increased their disulfide bond content. When MBPs with a 25% moisture content were heated at 85°C for 60 min, their gel properties notably improved, and their structural changes were maximal. The structural changes revealed that the MBP molecule formed a macromolecular polymer because a significant protein band appeared at about 66.2 kDa. Secondary structure and microstructure analyses revealed that the MBP structure was significantly damaged and that the β-sheet structure increased because of changes in the degree of aggregation between the protein molecules. Compared to the untreated MBPs, the MMBPs significantly improved the cooking quality and texture properties of the noodles, and the addition amount reached more than 6%, whereas that of the untreated MBPs was less than 3%. At this time, the cooking loss and the broken rate of the untreated MBPs group were about 2 times higher than that of the 6% MMBP-treated group. An analysis of changes in the water distribution, rheological properties, and microstructure revealed that intermolecular cross-linking occurred between the MMBPs and wheat dough, which improved the quality of the MMBP-treated noodles. The findings demonstrated that the MMBPs obtained by hydrothermal treatment had a positive effect on the wheat dough properties and noodle quality. These results provide a technical foundation for incorporating novel protein supplements into staple foods.

Three-dimensional tailor-made collagen-like proteins hydrogel for tissue engineering applications.

Despite the potential tunable properties of blank slate collagen-like proteins (CLP), an alternative to animal-originated collagen, assembling them into a stable 3D hydrogel to mimic extracellular matrix is a challenge. To address this constraint, the CLP (without hydroxyproline, CLPpro) and its variants encoding functional unnatural amino acids such as hydroxyproline (CLPhyp) and 3,4-dihydroxyphenylalanine (CLPdopa) were generated through genetic code engineering for 3D hydrogel development. The CLPhyp and CLPdopa were chosen to enhance the intermolecular hydrogen bond interaction through additional hydroxyl moiety and thereby facilitate the self-assembly into a fibrillar network of the hydrogel. Hydrogelation was induced through genipin as a cross-linker, enabling intermolecular cross-linking to form a hydrogel. Spectroscopic and rheological analyses confirmed that CLPpro and its variants maintained native triple-helical structure, which is necessary for its function, and viscoelastic nature of the hydrogels, respectively. Unlike CLPpro, the varients (CLPhyp and CLPdopa) increased pore size formation in the hydrogel scaffold, facilitating 3T3 fibroblast cell interactions. DSC analysis indicated that the stability of the hydrogels got increased upon the genetic incorporation of hydroxyproline (CLPhyp) and dopa (CLPdopa) in CLPpro. In addition, CLPdopa hydrogel was found to be relatively stable against collagenase enzyme compared to CLPpro and CLPhyp. It is the first report on 3D biocompatible hydrogel preparation by tailoring CLP sequence with non-natural amino acids. These next-generation tunable CLP hydrogels open a new venue to design synthetic protein-based biocompatible 3D biomaterials for tissue engineering applications.