Search Results (187)

The enzymatic production of low-molecular-weight chitosan and chitooligosaccharides (COS), with broad application potential in agriculture, food, medicine, and cosmetics, has emerged as an attractive alternative to chemical chitosan depolymerization owing to its substrate specificity and environmentally benign catalytic action. However, the functional properties of available chitosanases need to be enhanced to meet the demands of industrial COS manufacturing under high temperature and substrate concentrations. In this work, we performed directed evolution on a recombinant Bacillus subtilis chitosanase to increase chitosan hydrolysis performance and thermal resistance. Three rounds of directed evolution screening (~9000 clones) yielded variants MT1, MT2, and MT3 with higher specific activity, achieved through Vmax improvement and increased T_1/2 at 60 °C. HPLC, DLS, and MALDI-TOF results indicate differences in the hydrolysis kinetics and size distribution of COS products over reaction time, suggesting a narrower distribution and a lower average molecular weight. Molecular dynamics simulations and docking studies revealed potential modulation of chitosanase activity via changes in the opening and closing dynamics of the active-site cleft. These results suggest that future efforts targeting the cleft interface could significantly advance both the catalytic performance and the mechanistic understanding of GH46 family chitosanases. Full article

The influence of different gas compositions in modified atmospheric packaging (MAP) without and with chitooligosaccharide-EGCG (CE) conjugate on storage stability of Asian hard clam (HC) meat during storage at 4 °C was studied. Microbial load of HC meat was <5 log CFU/g when packaged under MAP, regardless of treatment, up to 18 days of storage, whereas control exceeded viable bacterial count (6 log CFU/g) on day 9. The lowest microbial load, volatile bases, and lipid oxidation were obtained in HC meat pretreated with 600 ppm of CE conjugate and MAP (80% CO₂/20% O₂) (MAP4-CE) (p < 0.05). Correlation heatmap analysis showed that a high-CO₂/low-O₂ atmosphere was the primary determinant of reduced Pseudomonas growth and lipid oxidation in HC meat, whereas the CE conjugate conferred only minor oxidation and nitrogenous spoilage indices. HC packed under MAP exhibited higher cooking and drip loss, along with increased toughness and firmness, irrespective of treatment. PUFA of MAP4-CE was retained during 18 days of storage. High-CO₂, with or without CE, redirected the microbial diversity toward CO₂-tolerant taxa. Overall, MAP4-CE had an extended shelf-life of at least 18 days while better preserving lipid quality and delayed growth of spoilage bacteria. Full article

The increasing demand for sustainable agriculture necessitates the development of eco-friendly alternatives to chemical pesticides. This study reports the design and characterization of biodegradable fibrous mats for the delivery of Bacillus subtilis, a plant-beneficial biocontrol agent, using cellulose acetate (CA) scaffolds functionalized with chitooligosaccharides (COS). Electrospun CA fibers were coated by electrospraying with COS or COS/B. subtilis suspensions in a single-step process to produce open, porous biohybrid scaffolds. Scanning electron microscopy confirmed uniform fiber formation and successful deposition of COS and bacterial layers, while ATR-FTIR spectroscopy verified the chemical composition of the fibrous mats. Water contact angle measurements indicated a shift from hydrophobic to highly hydrophilic surfaces, enhancing microbial adhesion and moisture-mediated activation. Mechanical testing demonstrated that thin COS coatings slightly improved tensile strength without compromising flexibility. Viability assays confirmed that encapsulated B. subtilis remained viable and capable of sporulation, and dual-culture assays demonstrated effective inhibition of Alternaria solani, Fusarium avenaceum, and Rhizoctonia solani. These results indicate that the electrospun/electrosprayed CA/COS platform provides a protective, sustainable, and effective delivery system for biocontrol agents. This approach offers a promising strategy for reducing reliance on synthetic pesticides while maintaining crop protection efficacy. Full article

Peppers are of substantial economic importance and hold a prominent position among vegetables rich in health-promoting compounds, which drives continuous efforts to develop improved cultivation strategies. The study aimed to determine the effects of substrate type and depolymerized chitosan on the physiological parameters, the chemical composition of leaves and fruits, and the yield of two bell pepper cultivars: ‘Marta Polka’ and ‘Oda’. The plants were grown in a 100% peat substrate and in a mixture of peat, wood fiber (Pinus sylvestris), and green compost (2:1:1 v/v/v), with or without drenching with a solution of depolymerized chitosan. Results indicated that the growing medium, chitosan application, cultivar type, and their interactions altered several physiological, morphological, and biochemical traits. The highest total fruit weight fresh (471.23 g plant⁻¹) was obtained for the ‘Marta Polka’ cultivar grown in peat drenched with chitosan, whereas the lowest (192.02 g plant⁻¹) was recorded for ‘Oda’ grown in a substrate mix without the biostimulant. Net CO₂ assimilation rate, stomatal conductance, fresh weight of fruit, and antioxidant activity (ABTS and FRAP assays) were improved in the ‘Oda’ cultivar grown in the substrate mix and treated with depolymerized chitosan compared with plants grown in 100% peat without chitosan. The ‘Marta Polka’ plants grown in the substrate mix and treated with chitosan had a higher net CO₂ assimilation rate, photosynthetic water-use efficiency, total free amino acid content, and antioxidant activity (FRAP assay) than those grown in peat alone and not treated with the biostimulant. The results demonstrate that both substrate composition and the response to depolymerized chitosan are cultivar-specific, and that wood fiber and compost can serve as ecological alternatives to peat, enhancing overall pepper fruit quality. Full article

Spermatogonial stem cells (SSCs) are the only adult male germline stem cells capable of lifelong self-renewal and differentiation into spermatozoa. Scalable ex vivo survival is essential for endangered species germplasm banking, genetic resource conservation, and male infertility therapy. Here, chitosan (CO) or chitosan oligosaccharide (COS) was cross-linked into injectable, biodegradable 3D hydrogels loaded with the natural astaxanthin (AST). CCK-8 optimization identified 0.3% CO + 0.2% AST (CHAG) and 0.2% COS + 0.2% AST (COAG) as superior formulations. After 7 or 14 d of 3D culture, CHAG yielded significantly more colonies than controls (p < 0.01), with elevated EdU incorporation, alkaline phosphatase activity, and positive OCT4 and PLZF staining, confirming preserved stemness. Caspase-3 expression was markedly reduced, indicating the AST-mediated suppression of oxidative apoptosis. RNA-seq showed distinct transcriptome pathways (p < 0.01): CHAG up-regulated adhesion and ECM–receptor and cell cycle pathways, whereas COAG enriched immune-modulatory and signaling modules, enabling context-specific use. AST-loaded CO/COS hydrogels are inexpensive, cytocompatible, and scalable, doubling as a biomimetic niche that accelerates SSC proliferation while delaying senescence. The platform provides a robust, controllable 3D system for SSC expansion and establishes a pre-clinical basis for translating CO/COS/AST composites to reproductive stem cell biotechnology. Full article

Protein-based APIs represent a big group of modern therapeutics. Their characterization involves complex analytical protocols which require special methods, especially in the case when the protein drug is included into tablet dosage forms. Although the fluorescence polarization assay (FPA) is not currently regulated by many national Pharmacopeias, it represents a promising approach for protein drug standardization, considering their rapid, sensitive, and automatable detection suitable for high-throughput analysis and real-time quality control. To evaluate the applicability of FPA for the analysis of protein drugs in tablets, the quantifying of lysozyme in tablet dosage forms was studied by this method with the use of a fluorescently labeled synthetic chitooligosaccharide tracer. It was shown that this approach overcomes the limitations of the conventional turbidimetric assay of lysozyme determination, which is labor-intensive and relies on unstable reagents. Measurements were performed with both portable and stationary fluorescence polarization readers. Commercial tablets from five manufacturers containing lysozyme (20 mg) and pyridoxine hydrochloride (10 mg) together with other excipients were analyzed. The FPIA method showed a linear range of 5.0–70 µg/mL, with specificity confirmed by the absence of interference from excipients. Accuracy, evaluated by standard addition (10–20 mg), yielded recoveries of 100.2–106.0%. Placebo spiked with lysozyme at 80–120% of nominal content demonstrated recoveries of 98.0–100.1%, with RSD (n = 6) not exceeding 13.7%, indicating good precision. The developed method enables reliable lysozyme quantification in tablets, offering speed, simplicity, and robustness, and shows its suitability for the routine quality control of protein-containing dosage forms including the enzyme ones. Full article

The growing need for sustainable protein and functional food ingredients has made edible insects stand out as a flexible source of bioactives. Black Soldier Fly larva (BSFL) bioactives, such as chitooligosaccharides (COSs) and peptides, present potential benefits for gut health; nevertheless, their molecular pathways, clinical validation, and commercial scalability have yet to be thoroughly investigated. This study systematically analyzes current progress in BSFL bioactive extraction and characterization, emphasizing enzymatic and thermal processing, controlled enzyme development, and integrated supercritical fluid enzymatic pipelines. We assess preclinical and animal research that illustrates prebiotic modulation of Bifidobacterium, Lactobacillus, and Faecalibacterium populations; antimicrobial peptide-mediated immune signaling; and antioxidant activity. Multi-omics frameworks that connect the microbial metabolism of COS to gut health help us understand how these processes function. A comparison of the regulatory environments for food and feed applications in the EU, North America, and Asia shows that there are gaps in human safety trials, harmonized standards, and techno-economic assessments. Finally, we suggest some next steps: randomized controlled human trials in groups with irritable bowel syndrome (IBS) and metabolic syndrome; standardized data integration pipelines for multi-omics; and life cycle and cost–benefit analyses of modular, vertically integrated BSFL biorefineries with AI-driven reactors, digital twins, and blockchain traceability. Addressing these issues will hasten the conversion of BSFL bioactives into safe, effective, and sustainable functional meals and nutraceuticals. Full article

Elderberry (Sambucus nigra L.) is recognized as a rich source of anthocyanins and other bioactives with antioxidant and antidiabetic potential, and is increasingly explored as a functional ingredient in nutraceuticals. However, cultivar-dependent variability can strongly influence chemical composition and bioactivity, underscoring the need for careful selection of plant material prior to formulation. In the present study, twelve genotypes of elderberry were compared in terms of total polyphenols, antioxidant activity, and antiglycation potential. Based on the overall profile, ‘Samyl 1’ was advanced to formulation trials. Spray-dried carrier systems were produced using galactooligosaccharides (GOS) or chitooligosaccharides (COS), with or without colloidal silica. GOS-based powders retained anthocyanins at levels approaching theoretical values and exhibited superior thermal stability, as evidenced by differential scanning calorimetry, thermogravimetric analysis, and degradation-kinetic modeling, whereas COS matrices provided less effective stabilization. Incorporation of silica significantly enhanced technological properties, improving recovery, reducing agglomeration, and increasing flowability, without compromising anthocyanin content. All powders displayed low moisture (2.5–7.1%), favorable morphology, and preserved functional activity, aligning with stability requirements for shelf-stable plant extracts. Overall, the study demonstrates that strategic cultivar selection combined with GOS–silica carrier systems enables the production of stable elderberry powders that maintain high anthocyanin content and bioactivity. Such multifunctional ingredients couple prebiotic functionality with efficient delivery of polyphenols, highlighting their potential in nutraceutical and pharmaceutical formulations. Full article

Chitosanases are glycoside hydrolases (GHs) that catalyze the endo- or exo-type cleavage of β-1,4-glycosidic linkages in chitosan, enabling the selective production of chitooligosaccharides (COSs) with well-defined structures and diverse bioactivities. Owing to their substrate specificity and environmentally friendly catalytic action, chitosanases have garnered increasing attention as sustainable biocatalysts for COS production, with broad application potential in agriculture, food, medicine, and cosmetics. This review provides a comprehensive overview of recent advances in chitosanase research, focusing on the catalytic mechanisms and structure–function relationships that govern substrate selectivity and functional divergence across different GH families. Microbial diversity and heterologous expression systems for chitosanase production are discussed in parallel with biochemical characterization to support the rational selection of enzymes for specific biotechnological applications. Advances in protein engineering and computational approaches are highlighted as strategies to improve catalytic efficiency, substrate range, and stability. In addition, bioprocess optimization is addressed, with emphasis on fermentation using low-cost substrates and the application of immobilized enzymes and nano-biocatalyst systems for green and efficient COS production. Summarizing and discussing previous findings are essential to support future research and facilitate the development of next-generation chitosanases for sustainable industrial use. Full article

Lytic polysaccharide monooxygenases (LPMOs) represent copper-dependent enzymes pivotal in breaking down resilient polysaccharides like cellulose and chitin by means of oxidation, creating more accessible sites for glycoside hydrolases. To elevate the conversion efficiency of chitin, an AA10 LPMO was identified from the genome of Saccharophagus degradans 2-40^T and heterologously expressed. The optimal pH for the activity of recombinant SdLPMO10A is 9.0, and the optimal temperature is 60 °C. Assessment of SdLPMO10A’s synergism with commercial chitinase indicated that when comparing the enzyme combination’s activity to the activity of chitinase alone, the synergistic effect was significant, and a one-pot reaction appeared superior to a two-step reaction. This discovery of a functional AA10 family LPMO presents a promising avenue for developing highly efficient catalysts for biomass conversion of chitin-rich food processing waste (e.g., shrimp shells) into bioactive chitooligosaccharides with applications in functional foods, such as prebiotics and antioxidants. Full article

Amelanchier alnifolia is a plant known for its nutritional and bioactive properties. Its leaves contain a high concentration of active compounds with significant antioxidant and antidiabetic effects, including strong α-glucosidase inhibitory potential. The combination of these bioactive leaf extracts with prebiotic substances, such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), and chitooligosaccharides (COS), enables the development of functional systems with enhanced beneficial properties. In this study, process optimization for leaves extraction was performed using a Plackett–Burman screening design, which identified key parameters for further optimization using the Box–Behnken design. The optimal extraction conditions were determined as follows: methanol content 58.06%, solid-to-solvent ratio 26.03 m/v, and extraction time 73.56 min. These conditions yielded the highest the total phenolic content (TPC). A comparative analysis of different cultivars revealed significant variations in polyphenol content among them. The formulated lyophilized systems with GOS, FOS and COS positively influenced the chlorogenic acid release profile, while maintaining the extract’s antidiabetic and antioxidant properties. FT-IR analysis confirmed the molecular interactions responsible for these effects. The prebiotic effectiveness of the systems was quantitatively evaluated using two key parameters: the prebiotic index (PI), and the prebiotic activity score (PAS). Microbiological analyses demonstrated the beneficial effects of prebiotic-enriched systems characterized by better prebiotic action on Bifidobacterium strains than the pure extract. These findings suggest that A. alnifolia leaf extracts, in combination with prebiotics, could serve as promising functional ingredients with potential applications in health-promoting and antidiabetic formulations. Full article

Vibrio parahaemolyticus is the leading cause of bacterial diarrhea in humans from shellfish consumption. In Thailand, blood clam is a popular shellfish, but homemade cooking often results in insufficient heating. Therefore, consumers may suffer from food poisoning due to Vibrio infection. This study aimed to determine the effect of chitooligosaccharide conjugated with epigallocatechin gallate (COS-EGCG) at different concentrations (200 and 400 ppm) combined with high-voltage atmospheric cold plasma (HVACP) on inhibiting V. parahaemolyticus in vitro and in challenged blood clam meat. Firstly, HVACP conditions were optimized for gas composition and treatment time (20 and 60 s); a 70% Ar and 30% O₂ gas mixture resulted in the highest ozone formation and a treatment time of 60 s was used for further study. COS-EGCG conjugate at 400 ppm with HVACP (ACP-CE400) completely killed V. parahaemolyticus after incubation at 37 °C for 6 h. Furthermore, an antibacterial ability of ACP-CE400 treatment against bacterial cells was advocated due to the increased cell membrane damage, permeability, and leakage of proteins and nucleic acids. Scanning electron microscopy (SEM) showed cell elongation and pore formation, while confocal microscopy revealed disrupted biofilm formation. Additionally, the shelf life of challenged blood clam meat treated with ACP-CE400 was extended to nine days. SEM analysis revealed damaged bacterial cells on the meat surface after ACP-CE400 treatment, indicating the antibacterial activity of the combined treatment. Thus, HVACP combined with COS-EGCG conjugate, especially at a highest concentration (400 ppm), effectively inhibited microbial growth and extended the shelf life of contaminated blood clam meat. Full article

Chitooligosaccharides (COSs), degraded products of chitosan or chitin, are attracting growing interest owing to their low degree of polymerization (DP), high solubility, and prominent anti-inflammatory activity. However, the correlation between their structure and anti-inflammatory activities still needs to be explored. In this study, we use LPS-stimulated RAW 264.7 macrophages as an inflammatory model to systematically evaluate COS1–7 for their effects on inflammatory mediators and NF-κB signaling pathways. The results of Griess assay, ELISA, and real-time quantitative PCR show that COSs can inhibit the expression of NO, iNOS, and pro-inflammatory cytokines (IL-6, TNF-α, MCP-1 and IL-1β), thereby attenuating inflammatory signaling. Notably, chitohexaose (COS6) exhibits the most significant anti-inflammatory effect, reducing the mRNA levels of LPS-induced iNOS, IL-6, and IL-1β and the production of IL-6 and TNF-α by more than 50%. Transcriptome, western blotting, and real-time quantitative PCR analysis reveal that COSs can inhibit the activation of the NF-κB signal pathway by down-regulating TLR2 levels. Additionally, molecular docking confirms that COSs retard TLR2/4 dimerization and LPS recognition by TLR4, affecting downstream signaling cascades. In summary, this study provides a valuable insight into the potential anti-inflammatory mechanism of COSs and highlights the possible applications in human health promotion by modulating receptor-mediated signaling pathways. Full article

The depolymerization of chitosan using chitinolytic enzymes is one of the most promising approaches for the production of bioactive soluble chitooligosaccharides (COS) due to its high specificity, environmental safety, mild reaction conditions, and potential for development. However, the comparative efficacy of bacterial chitinases and chitosanases in terms of yield, solubility, and antimicrobial activity of produced COS remains understudied. In this work, chitinase (73 kDa) and chitosanase (40 kDa) from the strain Bacillus thuringiensis B-387 (Bt-387) were purified using various chromatographic techniques and compared by their action on chitosan (DD 85%). The molecular mass and structure of generated COS was determined using TLC, LC-ESI-MS, HP-SEC, and C¹³-NMR techniques. Chitosanase converted the polymer more rapidly to short COS (GlcN₂-GlcN₄), than chitinase, and was more specific in its action on mixed bonds between GlcN and GlcNAc. Chitosanase needed a noticeably shorter incubation time and enzyme–substrate ratio than chitinase for production of larger oligomeric molecules (Mw 2.4–66.5 and 15.4–77.7 kDa, respectively) during controlled depolymerization of chitosan. Moreover, chitosanase-generated oligomers demonstrate better solubility and a higher antifungal activity in vitro against the tested plant pathogenic fungi. These features, as well as the high enzyme production and its simplified purification protocol, make chitosanase B-387 more suitable for the production of antifungal chitooligomers than chitinase. Full article

Fresh wet vermicelli is highly susceptible to microbial contamination during storage as a result of its high moisture content and rich nutrients, which leads to spoilage and deterioration. In addition to exerting a great impact on the quality of the product, this results in significant economic losses and potential food safety risks. This work aimed to identify spoilage microorganisms via traditional culturing methods and molecular biology techniques. The effects of environmental factors such as temperature and pH on the growth and development of the dominant spoilage fungi were investigated, and the inhibitory effects of both chemical (potassium sorbate) and natural antimicrobial agents (chitooligosaccharides, chitosan, tea polyphenols, citric acid, and ε-polylysine hydrochloride) were evaluated. The results indicated that Penicillium crustosum was the major spoilage microorganism in fresh wet vermicelli, whose optimal growth temperature and pH was 28 °C and 7, respectively. While conidial germination began at 7 h, hyphal formation was only observed after 12 h. Moreover, the findings suggest that both natural and chemical antimicrobial agents can effectively inhibit the growth of P. crustosum, with ε-polylysine hydrochloride being the strongest antimicrobial agent. Overall, the findings of this study provide a scientific foundation for improving the preservation of fresh wet vermicelli, which is of great significance for extending its shelf life and enhancing food safety. Full article