Search Results (78)

Rice (Oryza sativa L.), a staple food for over half of the global population, plays a pivotal role in food security. Among its two primary groups, japonica and indica, temperate japonica varieties are particularly valued for their high-quality grain and culinary uses. Although some of these varieties are adapted to cooler climates, they often suffer from reduced productivity or increased disease susceptibility when cultivated in warmer productive environments. These limitations underscore the need for breeding programs to incorporate biotechnological tools that can enhance the adaptability and resilience of the plants. However, New Genomic Techniques (NGTs), including CRISPR-Cas9, require robust in vitro systems, which are still underdeveloped for temperate japonica genotypes. In this study, we developed a reproducible and adaptable protocol for protoplast isolation and regeneration from the temperate japonica cultivars ‘Ónix’ and ‘Platino’ using somatic embryos as the starting tissue. Protoplasts were isolated via enzymatic digestion (1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10) in 0.6 M AA medium over 18–20 h at 28 °C. Regeneration was achieved through encapsulation in alginate beads and coculture with feeder extracts in 2N6 medium, leading to embryogenic callus formation within 35 days. Seedlings were regenerated in N6R and N6F media and acclimatized under greenhouse conditions within three months. The isolated protoplast quality displayed viability rates of 70–99% within 48 h and supported transient PEG-mediated transfection with GFP. Additionally, the transient expression of a gene editing CRISPR-Cas9 construct targeting the DROUGHT AND SALT TOLERANCE (OsDST) gene confirmed genome editing capability. This protocol offers a scalable and genotype-adaptable system for protoplast-based regeneration and gene editing in temperate japonica rice, supporting the application of NGTs in the breeding of cold-adapted cultivars. Full article

This study investigates the enhancement of nitrogen removal performance in an intermittent biological sponge iron system (BSIS) through the immobilization of aerobic denitrifying bacteria. The aim is to improve the efficiency of simultaneous nitrification and denitrification (SND) in the BSIS by optimizing the microbial community involved in nitrogen conversion. The immobilization technique not only stabilizes the microbial activity and abundance of aerobic denitrifying bacteria, but also promotes a more efficient denitrification process. The optimal material ratio of polyvinyl alcohol–sodium alginate gel beads was determined as 10 g/100 mL PVA, 4 g/100 mL SA, 2 g/100 mL CaCl₂, and 2 g/100 mL of bacterial suspension, achieving a maximum NO₃⁻-N removal rate of 91.73%. A response surface model (RSM), established for the operational conditions, (shaker speed, temperature, and pH) showed a high fitting degree (R2 = 0.9960) and predicted the optimal conditions for maximum NO₃⁻-N removal as 109.24 rpm, 23.6 °C, and pH 7.9. Compared to R1 (47.82%), R3 achieved a higher average total nitrogen (TN) removal rate of 95.49%, following the addition of immobilized aerobic denitrifying bacteria to the BSIS. Full article

This study investigated the effects of combining sodium alginate (ALG) with various natural hydrocolloids on the microstructure and release behaviors of microbeads. The encapsulation solutions were prepared at a 1:1 (w/w) ratio with ALG as the control and carrageenan (CAR), locust bean gum (LBG), acacia gum (ACA), pectin (PEC), and carboxymethyl cellulose (CMC) as experimental groups. Each formulation contained 0.2% (w/v) tartrazine and was extruded into a CaCl₂ solution for bead production. Encapsulation efficiency varied across formulations, with the lowest in the control (ALG-ALG) and highest in ALG-CAR and ALG-CMC, reaching 74% and 78%, respectively. The microbead sizes ranged from 2.07 to 3.48 mm, with the lowest particle diameter observed in ALG-ACA composites. Surface analysis showed smooth and uniform microbeads in the control (ALG-ALG), while ALG-LBG microbeads were rougher. Release kinetics were assessed using various models, with the Higuchi model best describing the release for most formulations (highest R² values). Tartrazine release followed pseudo-Fickian behavior in all formulations, with slower release in ALG-ACA and faster release in ALG-LBG microbeads. This study fills a gap in understanding how the incorporation of different natural hydrocolloids influences both the encapsulation efficiency and release dynamics of alginate-based microbeads, providing valuable insights for applications in food and pharmaceutical industries. Full article

This work describes the immobilization and the characterization of purified Penicillium crustosum Thom P22 lipase (PCrL) using adsorption, encapsulation, and adsorption–encapsulation approaches. The maximum activity of the immobilized PCrL on CaCO₃ microspheres and sodium alginate beads was shifted from 37 to 45 °C, compared with that of the free enzyme. When sodium alginate was coupled with zeolite or chitosan, the immobilization yield reached 100% and the immobilized PCrL showed improved stability over a wide temperature range, retaining all of its initial activity after a one-hour incubation at 60 °C. The immobilization of PCrL significantly improves its catalytic performance in organic solvents, its pH tolerance value, and its thermal stability. Interestingly, 95% and almost 50% of PCrL’s initial activity was retained after 6 and 12 cycles, respectively. The characteristics of all PCrL forms were analyzed by X-ray diffraction and scanning electron microscopy combined with energy dispersive spectroscopy. The maximum conversion efficiency of oleic acid and methanol to methyl esters (biodiesel), by PCrL immobilized on CaCO₃, was 65% after a 12 h incubation at 40 °C, while free PCrL generated only 30% conversion, under the same conditions. Full article

Adsorption is one of the most promising strategies for heavy metal removal. For Hg(II) removal, mineralized Ca-based shell-type self-assembly beads (MCABs) using alginate as organic polymer template were synthesized in this work. The adsorbent preparation consists of gelation of a Ca-based spherical polymer template (CAB) and rate-controlled self-assembly mineralization in bicarbonate solution with various concentrations. The comparative study demonstrates that 1% (MCAB-1) is the optimal concentration of bicarbonate. Based on this condition, the maximum adsorption capacity (48 ± 4 mg/g) of MCAB-1 was observed at pH = 5 in a batch test, which was 2.67 times more than that of the unmodified one, CAB, at 18 ± 1 mg/g. Long-duration (10 h) adsorption tests showed that MCAB-1 exhibited remarkable performance stability and anti-wear ability (43.2% removal efficiency and 74.3% mass retention, compared to 2.7% and 38.6% for CAB at pH = 3, respectively). The morphology determination showed that a shell-type porous amorphous carbonate layer was formed at the surface of the organic polymer template by rate-controlled self-assembly mineralization. This transition not only promotes the pore structure and activated cation binding functional sites, but also improves the anti-wear ability of materials effectively. Full article

Calcium alginate hydrogel is one of the most widely used materials for drug-carrier beads used in drug-delivery systems. In this study, we developed a new method to improve the encapsulation efficiency of ingredients, such as medicines, in calcium alginate hydrogel beads. In the gold standard method, the hydrogel beads are prepared in the liquid phase. In contrast, in the new method, to enhance the encapsulation efficiency, the hydrogel beads are prepared in the gas phase using a water-repellent surface. In brief, a droplet of sodium alginate aqueous solution is rolled on a water-repellent surface with CaCl₂ powder, a cross-linking agent. This process leads to the direct attachment of CaCl₂ powder to the droplet, resulting in the formation of spherical hydrogel beads with high mechanical strength and higher encapsulation efficiency than beads prepared by previous methods. The hydrogel beads exhibit similar permeability for glucose, a model for low-molecular-weight medicines, to those prepared by previous methods. These results show that the new method is promising for the preparation of calcium alginate hydrogel beads for drug-delivery systems. Full article

Chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile, TPN, CAS: 1897-45-6) is a halogenated fungicide currently widely applied to a large variety of crops. Its carcinogenicity, embryo lethality, and high chronic oral toxicity in mammals, among other effects on a variety of organisms, has made its biodegradation of great interest. Chlorothalonil dehalogenase (Chd) from the bacterium Pseudomonas sp. CTN-3 offers a potential solution by catalyzing the first step in the degradation of chlorothalonil. Reported herein are active biomaterials of Chd when encapsulated in tetramethylorthosilicate (TMOS) gels using the sol–gel method (Chd/sol), alginate beads (Chd/alginate), and chitosan-coated alginate beads (Chd/chitosan). Both Chd/sol and Chd/chitosan increased protection from the endopeptidase trypsin as well as imparted stability over a pH range from 5 to 9. Chd/sol outperformed Chd/alginate and Chd/chitosan in long-term storage and reuse experiments, retaining similar activity to soluble Chd stored under similar conditions. All three materials showed a level of increased thermostability, with Chd/sol retaining >60% activity up to 70 °C. All materials showed activity in 40% methanol, suggesting the possibility for organic solvents to improve TPN solubility. Overall, Chd/sol offers the best potential for bioremediation of TPN using Chd. Full article

Antioxidants derived from food by-products are known for their bioactive properties and impact on human health. However, the gastrointestinal behavior is often poor due to their degradation during digestion. The development of Ca(II)–alginate beads supplemented with biopolymers and enriched with cowpea (Vigna unguiculata) extract could represent a novel environmentally friendly technological solution to produce functional ingredients in the food industry. The present study evaluates the impact of in vitro digestion/fermentation by analyzing global antioxidant response (GAR), production of short-chain fatty acids (SCFAs) as a modulation of gut microbiota, and behavior of proton transverse relaxation times by low-field nuclear magnetic resonance (as an indicator of gelation state and characterization of microstructure). Results revealed that guar gum and cowpea protein preserved a high GAR of total phenolic compounds and antioxidant capacity by ABTS and FRAP methods after digestion/fermentation, promoting an adequate protection of the bioactives for their absorption. Alginate-based beads have great potential as prebiotics, with the guar gum-containing system contributing the most to SCFAs production. Finally, the overall higher mobility of protons observed in the intestinal phase agrees with structural changes that promote the release of phenolic compounds during this stage. Beads are excellent carriers of bioactive compounds (cowpea phenolic compounds and peptides) with potential capacities. Full article

The aim of this study was to produce and to characterize craft beer fermented by immobilized yeast cells with the addition of Prokupac grape pomace seed powder (2.5% and 5%), to obtain a beer enriched with phenolic compounds and improved sensory characteristics. The immobilization of the yeast cells was performed by electrostatic extrusion, while the obtained calcium alginate beads were characterized by light and scanning electron microscopy. Phenolic and hop-derived bitter compounds in beer with or without grape pomace seed powder (GS) phenolics were identified using UHPLC Q-ToF MS. The results indicated that GS adjunct significantly shortened the fermentation process of wort and increased the content of phenolic compounds, especially ellagic acid, flavan-3-ols and pro(antho)cyanidins in the final products compared to the control beer. A total of twenty (iso)-α-acids and one prenylflavonoid were identified, although their levels were significantly lower in beers with GS phenolics compared to the control beer. Beers with GS phenolics showed good antioxidant properties as measured by the reduction of ferric ions (FRP) and the scavenging of ABTS^•+ and DPPH^• radicals. The concentration of immobilized viable yeast cells was higher than 1 × 10⁸ CFU/g wet mass after each fermentation without destroying the beads, indicating that they can be reused for the repeated fermentation of wort. Beers produced with 5% GS added to the wort exhibited the best sensory properties (acidity, astringency, bitterness intensity, mouthfeel, aftertaste and taste), and highest overall acceptability by the panelists. The results showed that grape pomace seed powder present a promising adjunct for the production of innovative craft beer with good sensory properties and improved functionality. Full article

Carotenoids, the natural pigments that confer the bright orange color of sea buckthorn berries, are also associated with several health benefits, such as antioxidant activity and skin and eye protection. Due to their lipophilic nature and localization, carotenoids are largely retained in the sea buckthorn pomace (SBP) resulting from juice production. Carotenoids from SBP (70.03 mg/100 g DW), extracted and characterized by HPLC-PDA, contained zeaxanthin (free and esterified) and beta-carotene as major compounds. The SBP carotenoids-enriched sunflower oil was further encapsulated in Ca-alginate hydrogel beads (98.4% encapsulation efficiency) using ionotropic gelation. The hydrogel beads were characterized by confocal laser scanning microscopy and scanning electron microscopy. Fairly good stability (>64%) of the encapsulated carotenoids in the alginate hydrogel beads during storage (30 days, 4 °C and 25 °C) was found, with zeaxanthin esters being the most stable compounds, for all the experimental conditions. The bioaccessibility of the total carotenoids (INFOGEST protocol) was 42.1 ± 4.6% from hydrated, and, respectively, 40.8 ± 4% from dehydrated SBP alginate hydrogel beads. The addition of yogurt to the dehydrated hydrogel beads had a positive effect on the bioaccessibility of free and esterified zeaxanthin, but not on that of the carotenes. In conclusion, SBP is a valuable source of carotenoids which can be protected by encapsulation in alginate hydrogel beads, thus still retaining a good bioaccessibility. Full article

In this study, for enhancing the resistance of probiotics to environmental factors, we designed a microgel beads delivery system loaded with synbiotics. Multiple droplets of W₁/O/W₂ emulsions stabilized with zein–apple pectin hybrid nanoparticles (ZAHPs) acted as the inner “egg,” whereas a three-dimensional network of poly-L-lysine (PLL)-alginate-CaCl₂ (Ca) crosslinked gel layers served as the outermost “box.” ZAHPs with a mass ratio of 2:1 zein-to-apple pectin showed excellent wettability (three-phase contact angle = 89.88°). The results of the ζ-potentials and Fourier transform infrared spectroscopy demonstrate that electrostatic interaction forces and hydrogen bonding were the main forces involved in the formation of ZAHPs. On this basis, we prepared W₁/O/W₂ emulsions with other preparation parameters and observed their microstructures by optical microscopy and confocal laser scanning microscope. The multi-chambered structures of W₁/O/W₂ emulsions were successfully visualized. Finally, the W₁/O/W₂ emulsions were coated with PLL-alginate-Ca using the solution extrusion method. The results of the in vitro colonic digestion stage reveal that the survival rate of probiotics in the microgel beads was about 75.11%, which was significantly higher than that of the free. Moreover, probiotics encapsulated in microgel beads also showed positive storage stability. Apple pectin would serve as both an emulsifier and a prebiotic. Thus, the results indicate that the “egg-box” shaped microgel beads, designed on the basis of pH-sensitive and enzyme-triggered mechanisms, can enhance the efficiency of probiotics translocation in the digestive tract and mediate spatiotemporal controlled release. Full article

A novel organic–inorganic gliclazide-loaded composite bead was developed by an ionic gelation process using acidified CaCl₂, chitosan and tetraethylorthosilicate (TEOS) as a crosslinker. The beads were manufactured by crosslinking an inorganic silicone elastomer (-OH terminated polydimethylsiloxane, PDMS) with TEOS at different ratios before grafting onto an organic backbone (Na-alginate) using a 3² factorial experimental design. Gliclazide’s encapsulation efficiency (EE%) and drug release over 8 h (% DR 8 h) were set as dependent responses for the optimisation of a pharmaceutical formula (herein referred to as ‘G op’) by response surface methodology. EE % and %DR 8 h of G op were 93.48% ± 0.19 and 70.29% ± 0.18, respectively. G op exhibited a controlled release of gliclazide that follows the Korsmeyer–Peppas kinetic model (R² = 0.95) with super case II transport and pH-dependent swelling behaviour. In vitro testing of G op showed 92.17% ± 1.18 cell viability upon testing on C2C12 myoblasts, indicating the compatibility of this novel biomaterial platform with skeletal muscle drug delivery. Full article

There is a strong need to develop an insulin delivery system suitable for oral administration and preserving natural (α-helix) insulin conformation. In this work, we fabricated alginate–gelatin hydrogel beads for insulin encapsulation. Altering matrix composition and crosslinking agents has resulted in various surface morphologies and internal spatial organization. The structures of the insulin-loaded matrices were studied using optical and field emission electronic microscopy. We use FTIR spectroscopy to identify insulin conformation changes as affected by the hydrogel matrices. It was found that blended alginate–gelatin matrices demonstrate better encapsulation efficiency and stronger swelling resistance to a simulated gastric environment than sodium alginate beads crosslinked with the CaCl₂. FTIR measurements reveal conformation changes in insulin. It is also confirmed that in the presence of gelatin, the process of insulin fibrinogenesis ceases due to intermolecular interaction with the gelatin. Performed molecular modeling shows that dipole–dipole interactions are the dominating mechanism that determines insulin behavior within the fabricated matrix. Full article

The iminosugar 1-deoxynojirimicyn (DNJ) contained in mulberry leaves has displayed systemic beneficial effects against disorders of carbohydrate metabolism. Nevertheless, its effect is impaired by the short half-life. Alginate-based carriers were developed to encapsulate a DNJ-rich mulberry extract: Ca-alginate beads, obtained by external gelation, and spray-dried alginate microparticles (SDMs). Mean size and distribution, morphology, drug loading, encapsulation efficiency, experimental yield, and release characteristics were determined for the two formulations. Ca-alginate beads and SDMs exhibited an encapsulation efficiency of about 54% and 98%, respectively, and a DNJ loading in the range of 0.43–0.63 μg/mg. The in vitro release study demonstrated the carriers’ capability in controlling the DNJ release in acid and basic conditions (<50% in 5 h), due to electrostatic interactions, which were demonstrated by 1H-NMR relaxometry studies. Thus, alginate-based particles proved to be promising strategies for producing food supplements containing mulberry leaf extracts for the management of hyperglycemic state. Full article

In recent decades, there has been increased attention to the role of layer-by-layer assembled bio-polymer 3D structures (capsules, beads, and microgels) for biomedical applications. Such free-standing multilayer structures are formed via hard templating onto sacrificial cores such as vaterite CaCO₃ crystals. Immobilization of these structures onto solid surfaces (e.g., implants and catheters) opens the way for the formulation of advanced bio-coating with a patterned surface. However, the immobilization step is challenging. Multiple approaches based mainly on covalent binding have been developed to localize these multilayer 3D structures at the surface. This work reports a novel strategy to formulate multilayer surface-supported microgels (ss-MG) directly on the surface via hard templating onto ss-CaCO₃ pre-grown onto the surface via the direct mixing of Na₂CO₃ and CaCl₂ precursor solutions. ss-MGs were fabricated using biopolymers: polylysine (PLL) as polycation and three polyanions—hyaluronic acid (HA), heparin sulfate (HS), and alginate (ALG). ss-MG biodegradation was examined by employing the enzyme trypsin. Our studies indicate that the adhesion of the ss-MG to the surface and its formation yield directly correlate with the mobility of biopolymers in the ss-MG, which decreases in the sequence of ALG > HA > HS-based ss-MGs. The adhesion of HS-based ss-MGs is only possible via heating during their formation. Dextran-loading increases ss-MG formation yield while reducing ss-MG shrinking. ss-MGs with higher polymer mobility possess slower biodegradation rates, which is likely due to diffusion limitations for the enzyme in more compact annealed ss-MGs. These findings provide valuable insights into the mechanisms underlying the formation and biodegradation of surface-supported biopolymer structures. Full article