Development of Hypoallergenic Derivatives of Cra a 1 with B Cell Epitope Deletion and T Cell Epitope Retention.

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4+T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.

Structure, characterization, and application of a novel thermoreversible emulsion gel fabricated by citrate agar.

A novel thermoreversible emulsion gel was successfully prepared with citrate agar (CA) as the sole emulsifier. Compared with native agar gel emulsion, CA gel emulsion (CAGE) formed a stable emulsion gel when the CA concentration was increased to 1.25 % (w/w). Results of time-temperature scanning experiments showed that the emulsion gel rapidly transformed into liquid emulsion when heated to 40-50 °C and then solidified into emulsion gel after cooling to the critical temperature of solidification. The emulsion gel had stable sol-gel transformation ability after seven cycles repeated heating-cooling treatment (HCT) at 85 °C and 4 °C. However, the stability of emulsion gels gradually decreased because of the large-droplet formation during heating, which affected the CA molecular-reconfiguration network structure in cooling. The conjunction analysis of microstructure and properties of the emulsion gel indicated that its stability depended primarily on the spatial repulsion and electrostatic repulsion provided by CA gel, and the main factor driving thermal reversibility was the temperature-responsive gelation performance of CA. The retention of quercetin was >90.23 % after seven HCTs because CAGEG enhanced the homogeneity and stability of the droplets.

Agar-gelatin Maillard conjugates used for Pickering emulsion stabilization.

A few protein- and polysaccharide-based particles have shown promising potential as stabilizers in multi-phase food systems. By incorporating polymer-based particles and modifying the wettability of colloidal systems, it is possible to create particle-stabilized emulsions with excellent stability. A Pickering emulsifier (AGMs) with better emulsifying properties was obtained by the Maillard reaction between acid-hydrolysed agar and gelatin. Laser confocal microscopy imaging revealed that AGMs particles can be used as solid emulsifiers to produce a typical O/W Pickering emulsion, with AGMs adsorbing onto the droplet surface to form a dense interfacial layer. Cryo-scanning electron microscopy analysis showed that AGMs self-assembled into a three-dimensional network structure, which prevented droplets aggregation through strong spatial site resistance, contributing to emulsion stabilization. These emulsions exhibited stability within a pH range of 1 to 11, NaCl concentrations not exceeding 300 mM, and at temperatures below 80 °C. The most stable emulsion oil-water ratio was 6:4 at a particle concentration of 0.75 % (w/v). AGMs-stabilized Pickering emulsion was utilized to create a semi-solid mayonnaise as a replacement for hydrogenated oil. Rheological analysis demonstrated that low-fat mayonnaise stabilized with AGMs exhibited similar rheological behavior to traditional mayonnaise, offering new avenues for the application of Pickering emulsions in the food industry.

Fabrication, structure, characterization and emulsion application of citrate agar.

In this study, citric acid successfully reacted with agar through the dry heat method, and citrate agar (CA) gel was used to stabilize O/W emulsions. The mechanisms of the CA structure and emulsion pH that affected emulsion stabilization were analyzed, and the application of CA gel emulsion (CAGE) was explored. Compared with native agar (NA), CA showed lower gel strength, higher transparency, and higher water contact angle. These changes indicate that a cross-linking reaction occurred, and it was demonstrated via FTIR and NMR. The emulsion properties were evaluated using particle size, ζ-potential, and the emulsification activity index. Results showed that CAGEs had a smaller particle size and lower ζ-potential than the native agar gel emulsion (NAGE). Meanwhile, confocal laser scanning microscopy confirmed that the CA gels stabilized the emulsions by forming a protective film around the oil droplets. Stability experiments revealed that CAGE (prepared with CA gel [DS = 0.145]) exhibited better stability than NAGE in the pH range of 3-11, and the rheological results further confirmed that the stability of the emulsions was influenced by the network structure and oil droplet interaction forces. Afterward, the application prospect of CAGE was evaluated by encapsulating vitamin D3 and curcumin.

Efficient biosynthesis of prunin in methanol cosolvent system by an organic solvent-tolerant α-L-rhamnosidase from Spirochaeta thermophila.

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The Km of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while kcat/Km value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin.

Rational design of agarose/dextran composite microspheres with tunable core-shell microstructures for chromatographic application.

A new type of core-shell microsphere was prepared by a pre-crosslinking method, consisting of cross-linked agarose microspheres as the core and agarose-dextran as the shell. After optimizing the preparation process, the microspheres with a uniform particle size were obtained and characterized using cryo-scanning electron microscopy to determine their surface and cross-sectional morphology. Results from flow rate-pressure and chromatographic performance tests showed that the core-shell agarose microspheres were supported by the core microspheres and composed of composite polysaccharides, forming an interpenetrating polymer network structure as a hard shell. The core-shell agarose microspheres showed a 300.5 % increase in linear flow rate compared to composite polysaccharide microspheres prepared from shell materials and a 141.5 % increase compared to 6 % agarose microspheres. Additionally, the large pore structure of the shell combined with the fine pore structure of the core improved the material separation efficiency in the range of 0.1-2000 kDa. These findings suggest that core-shell natural polysaccharide microspheres have great potential as a separation chromatographic medium.

Bio-based active packaging: Gallic acid modified agarose coatings in grass carp (Ctenopharyngodon idellus) preservation.

Antioxidant and antimicrobial agarose coatings were developed by grafting gallic acid through the carbodiimide coupling method. Structural characterization revealed that the carboxyl group of gallic acid was successfully grafted onto the C6-OH of D-galactose in agarose, with the highest observed grafting ratio being 13.73 %. The grafting of gallic acid significantly increased the antioxidant and bacteriostatic activities of the agarose. As the grafting ratio of gallic acid-modified agarose (GaAg) increased from 0 to 13.73 %, the scavenging ratio of DPPH and the inhibition ratio of ß-carotene bleaching were observed to increase from 0 % to 65.92 % and 6.89 % to 73.46 %, respectively. GaAg exhibited up to 100 % inhibition of Escherichia coli and Staphylococcus aureus. The physicochemical properties of gel strength, viscosity, gelling temperature and melting temperature decreased to 971.3 g/cm2, 17.9 mPa·s, 31.7 °C and 84.1 °C, respectively. The gel contact angle was increased from 22.1° to 73.6°. Fish preservation tests have demonstrated that it effectively inhibited bacterial growth, prevented fat oxidation, blocked light, reduced moisture loss, and enhanced the overall quality of grass carp (Ctenopharyngodon idellus) fillets during refrigeration, which was more effective than native agarose in extending the shelf life of fish. Therefore, GaAg holds promise as an aquatic product preservative.

Preparation and Characterization of Carrageenase Immobilized onto Polyethyleneimine-Modified Pomelo Peel.

In this study, carrageenase immobilization was evaluated with a concise and efficient strategy. Pomelo peel cellulose (PPC) modified by polyethyleneimine (PEI) using the physical absorption method was used as a carrier to immobilize carrageenase and achieved repeated batch catalysis. In addition, various immobilization and reaction parameters were scrutinized to enhance the immobilization efficiency. Under the optimized conditions, the enzyme activity recovery rate was more than 50% and 4.1 times higher than immobilization with non-modified pomelo peels. The optimum temperature and pH of carrageenase after immobilization by PEI-modified pomelo peel, at 60°C and 7.5 respectively, were in line with the free enzyme. The temperature resistance was reduced, inconsistent with free enzyme, and pH resistance was increased. A significant loss of activity (46.8%) was observed after reusing it thrice under optimal reaction conditions. In terms of stability, the immobilized enzyme conserved 76.0% of the initial enzyme activity after 98 days of storage. Furthermore, a modest decrease in the kinetic constant (Km) value was observed, indicating the improved substrate affinity of the immobilized enzyme. Therefore, modified pomelo peel is a verified and promising enzyme immobilization system for the synthesis of inorganic solvents.

Media-milled agar particles as a novel emulsifier for food Pickering emulsion.

Pickering emulsions was successfully fabricated using ball-milled agar particles with sizes and sulfate content around 7 µm and 0.62 %, respectively. These particles were obtained through a simple media-milling process using agar powders initially sized at 120 µm. The lamellated agar is aggregated into a mass after the milling process. The surface charge and hydrophobicity of the ball-milled agar particles were characterized through zeta potential and contact angle measurements, respectively. The droplet size of Pickering emulsions was related to oil fraction and particle concentration, ranging from approximately 45 µm to 80 µm. Ball-milled agar stabilized emulsions were sensitive to pH and salt conditions. The results of confocal laser scanning microscopy and cryo-SEM showed that at low particle concentrations and oil fractions, ball-milled agar stabilized the emulsions by dispersing particles on the surface of the oil droplets through electrostatic repulsion. Conversely, ball-milled agar stabilized the emulsions under high particle concentrations and oil fractions by forming a gel network structure to bind the oil droplets. In this research, this developed method provides the basis for the high-value application of agar and a new idea for preparing stable food-grade Pickering emulsion-based functional foods using raw-food material without surface wettability.

Extraction, Modification and Biomedical Application of Agarose Hydrogels: A Review.

Numerous compounds present in the ocean are contributing to the development of the biomedical field. Agarose, a polysaccharide derived from marine red algae, plays a vital role in biomedical applications because of its reversible temperature-sensitive gelling behavior, excellent mechanical properties, and high biological activity. Natural agarose hydrogel has a single structural composition that prevents it from adapting to complex biological environments. Therefore, agarose can be developed into different forms through physical, biological, and chemical modifications, enabling it to perform optimally in different environments. Agarose biomaterials are being increasingly used for isolation, purification, drug delivery, and tissue engineering, but most are still far from clinical approval. This review classifies and discusses the preparation, modification, and biomedical applications of agarose, focusing on its applications in isolation and purification, wound dressings, drug delivery, tissue engineering, and 3D printing. In addition, it attempts to address the opportunities and challenges associated with the future development of agarose-based biomaterials in the biomedical field. It should help to rationalize the selection of the most suitable functionalized agarose hydrogels for specific applications in the biomedical industry.

Synthesis, characterization, antibacterial and emulsifying properties of agar benzoate.

Agar benzoate (AB) with different degrees of substitution (DS) was synthesized by the esterification of agar and benzoic anhydride in aqueous solution. The DS could be regulated by adjusting composition ratio, pH, and temperature. Its chemical structure was determined by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). 13C NMR spectrum of the AB suggested the main substitution occurred at the C-6 in the d-galactopyranose. Cryo-scanning electron microscopy (Cryo-SEM) showed that the aperture of AB was larger than that of agar. The thermal performance of AB decreased slightly, but this did not affect its performance. AB showed the highest relative antibacterial activity against Escherichia coli, S. aureus and Alternaria alternata, reaching 100 % (AB 20 g/L), 100 % (AB 40 g/L) and 19.35 % (7 d incubation), respectively. Moreover, the obtained AB possessed good emulsion stability. These antibacterial AB have broad application prospects in the field of fruit and vegetable preservation.

Effects of Lactobacillus fermentation on Eucheuma spinosum polysaccharides: Characterization and mast cell membrane stabilizing activity.

Eucheuma polysaccharides have varieties of biological activities. However, it is accompanied by problems like large molecular weight, high viscosity, and low utilization. Here, we first prepared fermented Eucheuma spinosum polysaccharides (F-ESP) by Lactobacillus fermentation, compared with low-temperature freeze-thaw ESP (L-ESP) prepared by the freeze-thaw method, explored the composition and structural characteristics of F-ESP and L-ESP, and evaluation of the ability of different samples to inhibit mast cell degranulation using classical mast cell model. Then, the activity of L-ESP and F-ESP in vivo was preliminarily evaluated using a passive cutaneous anaphylaxis model. Two kinds of F-ESP named F1-ESP-3 and F2-ESP-3 were obtained by fermentation of Eucheuma spinosum with the selected strains of Lactobacillus.sakei subsp.sakei and Lactobacillus.rhamnosus. Compared with the purified component L-ESP-3, the monosaccharide composition of F1-ESP-3 contains more glucuronic acid, the molecular weight reduced from >600 kDa (L-ESP-3) to 28.30 kDa (F1-ESP-3) and 33.58 kDa (F2-ESP-3), F1-ESP-3 has higher solubility and lower apparent viscosity. Fermentation did not destroy the functional groups and structure of ESP. Moreover, F1-ESP-3 significantly inhibited RBL-2H3 cell degranulation by reducing depolymerization of F-actin and Ca2+ influx. F1-ESP-3 reduced the symptoms of mast cell-mediated passive cutaneous anaphylaxis, indicating that F1-ESP-3 may have better anti-allergic activity in vivo.

Hydrophobic modified agar: Structural characterization and application in encapsulation and release of curcumin.

In this study, three kinds of anhydrides with different structures were introduced into agar molecules to study the effects of varying degrees of substitution (DS) and anhydride structures on the physicochemical properties and curcumin (CUR) loading capacity. Increasing the carbon chain length and saturation of the anhydride affects the hydrophobic interaction and hydrogen bonding of the esterified agar, thereby changing the stable structure of the agar. Although the gel performance declined, the hydrophilic carboxyl group and the loose porous structure provide more binding sites for the adsorption of water molecules, hence providing excellent water retention (1700 %). Next, CUR was used as a hydrophobic active ingredient to study agar microspheres' drug encapsulation and in vitro release ability. Results showed that the excellent swelling and hydrophobic structure of esterified agar could promote the encapsulation of CUR (70.3 %). The release process is controlled by pH, and the release of CUR under weak alkaline conditions is significant, which can be explained by the pore structure, swelling characteristics, and carboxyl binding of agar. Therefore, this study shows the application potential of hydrogel microspheres in loading hydrophobic active ingredients and sustained release and provides the possibility for the application of agar in drug delivery systems.

Super absorbent glutaric anhydride-modified agar: Structure, properties, and application in biomaterial delivery.

Agar was modified with glutaric anhydride (GA) in this study to expand its application in food and medicine. Glutaric anhydride-modified agar (GAR) can maintain high gel strength (1247.4 g/cm2) and improved transparency (82.7 %). The esterified agar formed by GA further formed a cross-linking molecule structure by increasing the reaction temperature. Notably, excellent freeze-thaw stability (24.1 %) and swelling property (3116.6 %) of GAR indicated that the carboxyl-terminal of modified agar improves its affinity with water. Therefore, satisfactory water permeability and expansive stone enable agar films to achieve high water absorption. Furthermore, GAR films exhibit a specific absorption capacity of tetracycline hydrochloride in weak acid solution, thereby suggesting its potential application as a sustainable drug delivery carrier. Finally, the structure of the modified agar was analyzed to explain the mechanism of binding water. Cryo-scanning electron microscopy (SEM) depicted the porous structure of the agar gel responsible for swelling, drug loading, and release. Low-field NMR results showed that GA improves agar gel's binding and free water content. According to our research results, these GAR hydrogel membranes with excellent properties have the potential to be used as effective drug delivery materials.

Effect of ß-glucosidase on the aroma of liquid-fermented black tea juice as an ingredient for tea-based beverages.

The effects of ß-glucosidase on the volatile profiles and aroma stability of black tea juice were evaluated using gas-chromatography-mass spectrometry coupled with sensory analysis. During liquid fermentation of tea leaves, the addition of ß-glucosidase increased the concentration of aldehydes, strengthening the undesirable "green grassy" odour. However, the "green grassy" odour was counteracted by adding green tea extract during fermentation. At the same time, "flowery" flavour notes were enhanced, improving the overall aroma quality and strengthening the characteristic "sweet" aroma of black tea. Increased addition of ß-glucosidase released more free aroma alcohols from their glucosides. Two "fruity" and "floral" aroma components, benzyl alcohol and phenylethyl alcohol, were not significantly affected by heat treatment (95 °C water bath) and the overall aroma stability was not significantly affected by ß-glucosidase treatment. ß-Glucosidase treatment improved the aroma, colour and overall suitability of fermented black tea juice as an ingredient for tea-based beverages.

A Novel κ-Carrageenase from Marine Bacterium Rhodopirellula sallentina SM41: Heterologous Expression, Biochemical Characterization and Salt-Tolerance Mechanism Investigation.

κ-carrageenases are members of the glycoside hydrolase family 16 (GH16) that hydrolyze sulfated galactans in red algae, known as κ-carrageenans. In this study, a novel κ-carrageenase gene from the marine bacterium Rhodopirellula sallentina SM41 (RsCgk) was discovered via the genome mining approach. There are currently no reports on κ-carrageenase from the Rhodopirellula genus, and RsCgk shares a low identity (less than 65%) with κ- carrageenase from other genera. The RsCgk was heterologously overexpressed in Escherichia coli BL21 and characterized for its enzymatic properties. RsCgk exhibited maximum activity at pH 7.0 and 40 °C, and 50% of its initial activity was retained after incubating at 30 °C for 2 h. More than 70% of its activity was maintained after incubation at pH 6.0-8.0 and 4 °C for 24 h. As a marine derived enzyme, RsCgk showed excellent salt tolerance, retaining full activity in 1.2 M NaCl, and the addition of NaCl greatly enhanced its thermal stability. Mass spectrometry analysis of the RsCgk hydrolysis products revealed that the enzyme had high degradation specificity and mainly produced κ-carrageenan disaccharide. Comparative molecular dynamics simulations revealed that the conformational changes of tunnel-forming loops under salt environments may cause the deactivation or stabilization of RsCgk. Our results demonstrated that RsCgk could be utilized as a potential tool enzyme for efficient production of κ-carrageenan oligosaccharides under high salt conditions.

A novel Pickering emulsion stabilized solely by hydrophobic agar microgels.

A novel agar-based Pickering emulsion stabilizer was developed through the hydrophobic modification and microgelation of agar. After hexanoylation, the three-phase contact angle of hexanoylated agar (HAG) particles was adjusted from approximately 60° to 96° closing to neutral wettability. After microgelation, the particle size of the modified agar microgel was approximately 2 µm and Zeta potential reached -23.63 mV. Confocal laser microscopy and cryogenic scanning electron microscopy showed that HAG microgels formed a gel network or embedded on the surface of oil droplets, thus providing a dense barrier for oil droplets to coalesce and Ostwald ripening. The oil volume fraction and particle concentration had a significant effect on the droplet size and rheological properties of the Pickering emulsion. Pickering emulsion gels with long-term storage stability was prepared at low particle concentrations (0.7 wt%) and lower oil fractions (φ = 0.3- 0.5), which might become a new effective delivery system for bioactive substances.

Preparation of macroporous rigid agarose microspheres by pre-crosslinking with cyclic anhydride.

In this study, a new method for preparing macroporous rigid agarose microspheres was developed by one-step pre-crosslinking method with cyclic anhydride. Three different cyclic anhydrides, namely, maleic anhydride, succinic anhydride, and glutaric anhydride, were used to pre-crosslink agarose. The reaction temperature and the amount of cyclic anhydride in the pre-crosslinking process were optimized to endow agarose with stronger cross-linking. Under the optimal cross-linking condition, macroporous rigid agarose microspheres with homogeneous particle size were successfully obtained by adjusting emulsification method. Cryo-scanning electron microscopy was used to characterize the morphology of cross-linked agarose gel and microspheres. The addition of cyclic anhydride increased the gel aperture of cross-linked agarose microspheres, thereby making the macropores in the microspheres more dense and enhancing the mass transfer in the particles. Under low pressure, the cross-linked agarose microsphere column can effectively separate model proteins at linear flow rates three times higher than the agarose microsphere column. These results indicate that the developed agarose microspheres are a promising high-speed chromatographic medium.

Eco-Friendly Extraction, Structure, and Gel Properties of ι-Carrageenan Extracted Using Ca(OH)2.

An eco-friendly method for ι-carrageenan extraction from seaweed Eucheuma denticulatum through boiling and using a low concentration of Ca(OH)2 is reported. Compared to the traditional method of ι-carrageenan extraction using NaOH, the reported method using Ca(OH)2 had the advantages of using 93.3% less alkali and 86.8% less water, having a 25.0% shorter total extraction time, a 17.6% higher yield, and a 43.3% higher gel strength of the product. In addition, we evaluated the gel properties and structures of ι-carrageenan products extracted by Ca(OH)2 (Ca-IC) and NaOH (Na-IC). The Fourier transform infrared spectroscopy results showed that the structures of Ca-IC and Na-IC did not change remarkably. The results of the thermogravimetric analysis and differential scanning calorimetry showed that Ca-IC had the same thermal stability as Na-IC. The results of the textural analysis showed that Ca-IC had a higher hardness and better chewiness compared to Na-IC. Rheological results indicated that Ca-IC and Na-IC exhibited shear-thinning and non-Newtonian fluid properties, whereas the viscosity of Ca-IC was less than that of Na-IC. In conclusion, this new method of ι-carrageenan extraction using Ca-IC is markedly better and yields higher quality carrageenan than the conventional method of using Na-IC.

Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement.

As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times kcat/Km as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process.