Small plant defense proteins known as nonspecific lipid transfer proteins (nsLTPs) possess diverse biological activities and promising therapeutic potential. Eleusine coracana (finger millet) is a nutrient-rich cereal crop recognized for its diverse bioactive compounds and potential health promoting properties. In this study, the proteins were extracted, dialyzed and four fractions were obtained by RP-HPLC. Further, the LC-MS/MS based peptide mass fingerprinting identified a bioactive nsLTP in the fraction 1. The pure nsLTP showed strong anti-quorum sensing activity against Pseudomonas aeruginosa and dual inhibitory activity against α-amylase and trypsin. At the minimum inhibitory concentration, the nsLTP reduced biofilm formation by 77 %, along with significant inhibition of exopolysaccharide, protease, elastase, and pyocyanin production. Confocal laser scanning microscopy confirmed biofilm disruption, while RT-qPCR analysis revealed the downregulation of quorum-sensing associated genes. Molecular docking further suggested favorable interactions of nsLTP with the quorum-sensing autoinducer 3-oxo-C12-HSL and the LasI, LasR, and LasA proteins, supporting its potential anti-quorum sensing mechanism. These results demonstrate the novelty of E. coracana derived nsLTP as a multifunctional bioactive protein with enzyme inhibitory and anti-virulence characteristics, indicating as a natural therapeutic candidate for managing P. aeruginosa infections and battling antibiotic resistance.
This study explored the microencapsulation of mandarin (Citrus reticulata) peel extract using ionic gelation and freeze-drying to improve the stability and bioavailability of citrus bioactives. Encapsulation involved sodium alginate, maltodextrin (MD), gum arabic (GA), and Aloe vera gel powder (0.3%), with a 20:80 core-to-wall ratio. Different formulations with varying MD and GA concentrations (total 2%) were tested for their effects on encapsulation efficiency, physicochemical properties, release behavior, and storage stability. Ionic gelation produced calcium-alginate beads with good biocompatibility but lower encapsulation efficiency; IG4 (1.5% MD, 0.5% GA) showed the best results in this group. Freeze-drying with ultrasonic homogenization significantly improved encapsulation efficiency, polyphenol retention, and antioxidant activity, with FD4 showing preferred formulation. Release studies revealed a biphasic, pH-dependent release profile, and MD-GA blends enabled controlled release, especially in FD4. Simulated gastrointestinal conditions showed higher release in the gastric phase, while FD4 and FD5 provided better protection and bioavailability. Storage stability followed first-order kinetics, with FD4 exhibiting the highest stability and longest shelf life. Overall, freeze-drying was more effective, thereby supporting the valorization of citrus waste to develop functional, antioxidant-rich food ingredients.
The study focused on the rational design of recombinant human G-CSF (rhG-CSF) by structure-based single-site modification and process optimization. The research was conducted in two main stages. In the first stage, a new A7G point mutation was introduced to the 3D structure of rhG-CSF by means of computational design. The mutant model, following energy minimization, displayed satisfactory stereochemistry (Molprobity clashscore 2.32) and maintained the native fold, suggesting that the mutation is structurally compatible. The second phase involved optimization of a high-yield expression system with the use of recombinant vector pET3a in E. coli BL21(DE3). Optimization of culture conditions showed that 4XYT medium with 0.5 mM IPTG and a 17-hour induction time resulted in the highest level of expression of the mutant protein. Such conditions led to a very high biomass yield of 96 grams of cells. The novelty of the present research work lies in the incorporation of the structure-guided single residue modification and process optimization in one step, resulting in structurally validated A7G rhG-CSF and process optimization to 5 L scale in complex medium.
Global fruit production reached 953.8 million tonnes in 2024, generating peel residues of 15%-50% of fruit weight that are largely discarded despite containing polyphenols, carotenoids, dietary fiber, and enzymes of commercial value. Existing reviews address this in fragments, covering extraction, green solvents, or food applications alone, but none traces the full pipeline from raw peel to finished product. This review provides an integrated, critically evaluated assessment of the entire valorization chain: extraction, purification, characterization, biological evaluation, and application. Rather than cataloguing methods, we compare them against yield, solvent use, energy demand, environmental impact, and scalability. Ultrasound- and microwave-assisted extraction cut processing time by 70%-95% and solvent use by 50%-90%, while supercritical CO2 extraction offers selectivity for non-polar compounds at high cost. Purification is the least standardized step, with resin adsorption and membrane separation most scalable. Characterization suffers from inconsistent validation, with detection limits, recovery, and matrix effects often unreported. Peel bioactives act through defined pathways (NF-κB, PI3K/AKT/mTOR, apoptosis, Nrf2, AMPK/GLUT4), yet efficacy is often shown at concentrations exceeding achievable plasma levels, making bioavailability the principal translational barrier. This review will be valuable to researchers in food science, analytical chemistry, and pharmaceutical sciences by integrating these traditionally different stages and identifying the key challenges that limit industrial and clinical translation.
Nisin is an antimicrobial peptide which offers potential for the replacement of synthetic preservatives in the food industry. However, nisin is difficult to produce on a large scale due to low synthesis efficiency and severe feedback inhibition. The objective of this work was to promote nisin production by integrating rare earth salt stimulation and foam separation with fermentation system. Experimental results indicated that the addition of 1 × 10-4 M TmCl3 into fermentation medium could significantly stimulate cell growth and nisin synthesis. The suitable feeding time of TmCl3 was the ninth hour after fermentation starting. Multiple characterization methods were used to explore the potential mechanism for TmCl3 stimulation. The increase in TmCl3 concentration contributed to improving the permeability of cell wall, but caused the degradation or denaturation of nucleic acids. Moreover, the addition of TmCl3 could regulate the expression of genes related precursor peptide synthesis and immunity. Finally, foam separation had been used for in-situ recovering nisin from the fermentation system. Under the optimum conditions predicted by using response surface methodology, the recovery percentage of nisin was 93.64 ± 1.31%. Compared with fermentation process without TmCl3 addition and foam separation, the total nisin titer of 8612.39 ± 140.58 IU/mL was increased by 114.44 ± 8.89%.
Dextran is a microbial homopolymer of α-glucan, consisting of α-(1,6) glycosidic linkage backbone with side chains branching via α-(1,2), α-(1,3), or α-(1,4) linkages. Due to its distinctive characteristics, including molecular mass, compatibility, and thermal stability, it is widely applied in various fields, such as food, pharmaceutical, biomedical, and environmental applications. Although previous reports have illustrated the application of microbial sources and the essential factors influencing fermentation and enzymatic pathways, a sustainable strategy for bioprocess engineering to produce microbial dextran has not been elaborated upon. This review provides a systematic and comprehensive overview of microbial dextran production, aiming to enable green bioprocess engineering strategies that enhance production for industrial applicability, promoting a sustainable approach.
Lignin is one of the most abundant yet recalcitrant biopolymers in nature, and its inefficient depolymerization remains a major bottleneck in lignocellulosic biomass valorization and sustainable biorefinery development. Although fungal oxidative enzymes have emerged as promising biocatalysts for lignin degradation, current knowledge remains fragmented across fungal diversity, catalytic mechanisms, enzyme engineering, and industrial translation. This review critically integrates recent advances in fungal ligninolytic systems, focusing on the structure, catalytic mechanisms, and synergistic interactions of major oxidative enzymes, including lignin peroxidase, manganese peroxidase, versatile peroxidase, laccase, and auxiliary oxidases such as aryl alcohol oxidase and glyoxal oxidase. Particular emphasis is placed on the comparative catalytic efficiency, operational limitations, and industrial feasibility of these enzymes in lignin depolymerization and biomass conversion. Recent developments in protein engineering, directed evolution, heterologous expression, immobilized enzyme systems, synthetic biology, and AI-assisted enzyme optimization are also discussed as emerging strategies to improve enzyme stability, substrate specificity, and process scalability. The review further highlights the role of fungal oxidative enzymes in bioremediation, lignocellulosic biorefineries, wastewater detoxification, biofuel production, and synthesis of value-added aromatic compounds. Importantly, it establishes a direct link between fungal ligninolytic systems and circular bioeconomy models through sustainable waste valorization and biomass-to-value conversion. Overall, this review provides an integrated perspective on the challenges, technological advancements, and future prospects of fungal oxidative enzymes for sustainable industrial biotechnology.
The textile industry generates large volumes of dye-laden effluents that pose risks to aquatic ecosystems and human health. This study evaluates the bioaccumulation of Reactive Black 5 (RB5) using Candida boidinii and Kluyveromyces marxianus yeast strains. Key operational parameters including initial pH, incubation time, dye concentration, and microbial growth were systematically investigated. In addition, the effects of low-cost agricultural wastes-carrot pomace (CP), industrial tea waste (ITW), and pumpkin pomace (PP)-as supplementary substrates were compared. Results showed that CP was the most effective substrate, with 100 g/L CP significantly enhancing RB5 removal. At pH 5.0 and 50 mg/L RB5, removal efficiencies reached 96.88% for C. boidinii and 81.76% for K. marxianus with CP, compared to 72.90% and 65.07% with PP. Although removal efficiency decreased at higher dye concentrations, the maximum dye uptake capacity (qm) increased with initial RB5 concentration, reaching 52.52 mg/g for C. boidinii and 33.23 mg/g for K. marxianus over the tested range (≈50-600 mg/L). To our knowledge, this is the first report demonstrating RB5 removal by these yeasts using CP as a low-cost substrate. Overall, CP significantly improves yeast-based RB5 bioaccumulation and represents a promising, sustainable option for treating dye-contaminated wastewater.
Plant polysaccharides have attracted considerable interest in natural product research due to their immunomodulatory, antitumor, and hypoglycemic activities. The development of efficient and gentle extraction and purification techniques is crucial for obtaining highly pure bioactive polysaccharides. This review systematically covers recent progress in extraction methods, including conventional hot water (simple but time‑consuming), acid/alkali (high yield but structure‑damaging), enzyme‑assisted, and physical field‑assisted techniques such as ultrasound and microwave, which provide higher efficiency under gentler conditions. For purification, a comparative analysis was conducted on the advantages and limitations of physical/chemical and biological methods for purifying polysaccharides. Furthermore, the review addresses current challenges, such as limited selectivity and throughput, and explores future directions, including the adoption of green solvents, intelligent process control, and integrated extraction-purification-evaluation platforms, with the objective of facilitating standardized production and high-value utilization.
L-Asparaginase is an important enzyme with therapeutic applications. Current commercial enzymes from E. coli and Erwinia chrysanthemi have limitations, such as high glutaminase activity, creating a need for more stable alternatives. This study aimed to isolate and optimize the production of L-Asparaginase from native, halotolerant Bacillus subtilis strains (DAR and D6A) isolated from central Iran, to find an alternative source for this therapeutically important enzyme. The enzyme was initially isolated using polyethylene glycol (PEG) precipitation. Key factors affecting enzyme production were screened using the Plackett-Burman design and subsequently optimized via the Box-Behnken response surface methodology. PEG precipitation at concentrations of 15-20% for PEG 4000/6000 and 10-15% for PEG 8000/10000 was most effective for initial enzyme separation, yielding specific activities of 4.8 U/mg and 5.2 U/mg for strains DAR and D6A, respectively. Optimization revealed that the most influential factors for maximum L-Asparaginase production were 17.6 g/L NaCl, 4.99 g/L KH2PO4, and 10 g/L asparagine for strain DAR, and 1.49 g/L glucose, 10 g/L NaCl, and 9.98 g/L asparagine for strain D6A. The isolated halotolerant B. subtilis strains are capable of producing L-Asparaginase. PEG precipitation is a suitable method for initial purification, and statistical optimization successfully identified the critical culture conditions to enhance enzyme yield significantly. These strains represent promising sources for the production of this clinically valuable enzyme.
Anaerobic biodegradation of bioplastics has gained increasing attention as a sustainable strategy for waste valorization and renewable energy recovery within circular bio-economy systems. The present study investigated the anaerobic biodegradation of Poly lactic acid (PLA) pellet, PLA film, Polybutylene succinate (PBS), and commercially available bioplastics under controlled conditions at different temperatures and incubation periods. Biodegradation behavior was evaluated through cumulative gas production, visual observations, FTIR characterization, kinetic modeling, and microbial analysis. All biopolymer samples exhibited significant cumulative gas production within 20 days before reaching the stationary phase. Among the biopolymer materials, PLA film showed distinct degradation behavior with high total solids (92.5%) and volatile solids (75.4%), along with the highest cumulative gas production. FTIR analysis and observable structural changes further confirmed polymer degradation during anaerobic digestion. Microbial species associated with biodegradation were identified through PCR amplification, 16S rRNA sequencing, and phylogenetic analysis, revealing the involvement of Bacillus sp in the degradation process. The cumulative biogas production profiles of all biopolymers followed the Hill sigmoidal kinetic model with high correlation coefficients (R2: 0.9854, 0.9839, 0.9931, and 0.9772 for PLA pellet, PBS, PLA film, and bioplastic, respectively), indicating excellent model fitting accuracy. Moreover, a generalized anaerobic biodegradation model equation was developed for biopolymers. The findings demonstrated the potential applicability of anaerobic digestion for biopolymer waste valorization through simultaneous biodegradation and biogas generation, supporting sustainable waste management and circular bio-economy approaches.
Biofilm technology is an effective biological wastewater treatment method that has attracted significant attention. As the core component of this technology, biocarriers play a vital role in microorganism attachment and biofilm formation. Polymer materials have long been favored as biocarrier materials due to their excellent properties. However, characteristics such as surface smoothness, hydrophilicity, and electronegativity can hinder microbial colonization and cause biofilms to detach easily. Therefore, developing new modified materials is a key strategy for improving the performance of traditional polymer-based biocarriers. This paper systematically reviews the research progress on modification strategies for polymer-based biocarrier materials, focusing on bulk modification (including filling, blending, and crosslinking), surface modification (such as grafting, etching, coating, and impregnation), and composite modification. Additionally, future research directions in this field are discussed. The goal is to advance the development of high-performance biocarrier materials.
In this study, the glucansucrase-encoding gene (dsr) from Weissella confusa P2 was cloned and heterologously expressed in Escherichia coli BL21(DE3). A 4542 bp fragment encoding 1510 amino acids was obtained, and the recombinant enzyme exhibited a molecular weight of approximately 161 kDa with a theoretical pI of 4.74. After Ni-IDA affinity chromatography, the specific activity reached 300.19 U/mg with a purification fold of 18.43. Enzymatic characterization revealed that the recombinant glucansucrase displayed optimal activity at pH 5.5 and 30 °C, and retained over 70% relative activity after 48 h incubation at 10-30 °C. The enzyme was significantly activated by Ca2+, while strongly inhibited by Ni2+, Cd2+, SDS, EDTA, DTT, and high concentrations of organic solvents. Kinetic analysis using sucrose as substrate yielded Km of 0.56 mM and Vmax of 4.57 U/mg, indicating exceptionally high substrate affinity. Using this recombinant enzyme, exopolysaccharide (EPS) was synthesized in vitro, achieving a yield of 59.23 ± 0.21 g/L, representing an 18.79% improvement over EPS synthesis by the wild-type strain. These findings enrich the genetic resources of glucansucrase, provide a promising biocatalyst for efficient EPS production, and establish a foundation for the application of glucansucrase and its polysaccharides products in the food and biotechnology industries.
Lactococcus lactis SH6 was engineered for heterologous heparosan production, and its growth medium was optimized using a combination of experimental design and machine learning (ML). One-factor-at-a-time shake flask experiments revealed glucose (10 g/L) and yeast extract (17.5 g/L) as the best substrates, producing 50 mg/L heparosan. Plackett-Burman analysis and steepest ascent optimization revealed significant factors, and a central composite design (CCD) optimized nutrient concentrations, predicting 85 mg/L heparosan (validated at 81 mg/L). ML-Gaussian process regression was applied after CCD optimization to fine-tune and cross-check the optimal medium (glucose 8.94 g/L, yeast extract 22.89 g/L, ascorbate 0.38 g/L, β-glycerophosphate 28.2 g/L), producing 85.28 mg/L heparosan (predicted 88.8 mg/L) at the flask scale. Earlier nisin induction (2 h) at the bioreactor scale increased heparosan titers to 119.7 mg/L, and linear glucose feeding (1.5 g/L.h) extended the production phase to 133 mg/L. Medium optimization resulted in nearly doubling heparosan yield compared to the unoptimized medium, setting a new standard for L. lactis. This work offers a design-of-experiments-ML solution as a viable approach to designing high-yielding, animal-product-free heparosan production methods in a Generally Regarded as Safe (GRAS) microbe.
Salmonella enterica serovar Typhi remains a major public health concern that requires an effective strategy to produce vaccines. This study reports the development of a high cell density fermentation (HCD) process to increase the production of Vi- polysaccharide (ViPS). The strain has been confirmed by means of phenotypic, biochemical (VITEK 2), and serological methods. Media optimization identified soybean peptone as a cost-effective and non-animal component alternative that promotes robust growth. The optimized fed-batch fermentation process increased the cell density from OD600 19-20 under low-cell-density conditions to OD600 39-42.1 under high-cell-density conditions, resulting in an approximately 1.7-fold increase in Vi polysaccharide (ViPS) yield i.e., from 165-187.5 mg/L to 283.75-302.5 mg/L. A scaling to 30 L fermentation tanks yielded comparable yields (290-320 mg/L), demonstrating the robustness of the process. Thermal inactivation at 65 °C for 30 min achieved complete cell inactivation without compromising the integrity of the polysaccharide, as predicted by the predictive model (R2 = 0.9947). NMR analysis confirms the structural integrity. The purified ViPS met WHO specifications, indicating suitability for cost-effective and scalable typhoid vaccine production.
In the present study, a native Bacillus subtilis isolate-8 was subjected to sequential mutagenesis using ultraviolet (UV) irradiation, nitrous acid (HNO2) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to obtain enhanced BNC production by following mutagenesis steps, with maximum yields of 4.3 g/L, 8.1 g/L and 9.8 g/L achieved by the superior UV mutant (BCUV-08), nitrous acid mutant (BCN-06), and NTG mutant (BCNT-03), respectively. The hyperproducing mutant BCNT-03 was further optimized using response surface methodology (RSM) based on a central composite rotatable design (CCRD). Four critical process variables, namely inoculum size (0.5-2.5%, v/v), incubation temperature (20-40 °C), incubation period (5-25 days), and flask-to-medium ratio (1:6-1:2, v/v), were evaluated under static fermentation conditions using modified Hestrin-Schramm (HS) medium containing molasses as a low-cost carbon source. The developed quadratic model was highly significant (F = 16.27, p < 0.001) and explained 93.44% of the variation in BNC production (R2=0.9344). The optimum conditions predicted by the model corresponded to an inoculum size of 1.5% (v/v), incubation temperature of 30 °C, incubation period of 15 days, and flask-to-medium ratio of 1:4, resulting in a maximum BNC yield of 15.92 g/L. The combined mutagenesis and process optimization strategy substantially enhanced BNC production. The findings demonstrate the potential of B. subtilis as an alternative BNC-producing organism.
Streptomyces pilosus is the primary producer of desferrioxamine B (DFOB), to treat acute and chronic iron overload. Its growth and productivity depend on dissolved oxygen concentration. Vitreoscilla hemoglobin (VHb enhances oxygen transfer and metabolic efficiency under oxygen-limited conditions. In this study, the vgb gene was cloned into S. pilosus to enhance DFOB production. The shuttle vector pGM1192 was designed in SnapGene, synthesized, and propagated in E. coli TOP10. PCR and restriction digestion with NdeI and BamHI confirmed recombinant plasmids. The recombinant plasmid was subsequently transferred into S. pilosus via electroporation. Transformation was verified through colony PCR and electrophoresis on a 1% agarose gel. The kinetics of cell growth and DFOB production in the recombinant strain expressing VHb were analyzed and compared with those of the wild-type strain. The recombinant VHb-expressing strain showed improved growth and DFOB production relative to the wild-type strain, achieving an OD600 of approximately 11 and producing about 1.2 g/L DFOB within 24 h. In contrast, under identical conditions, the wild-type strain reached only an OD600 of about 5.5 and produced approximately 0.4 g/L DFOB. These results indicate that VHb-mediated oxygen enhancement represents an effective metabolic engineering strategy for boosting DFOB biosynthesis in S. pilosus.
This study shows the effect of Zinc oxide nanorods, synthesized using Moringa oleifera phytochemicals as reducing and stabilizing agents were synthesized via a green method and systematically characterized to establish a consistent dataset linking structure, properties, and photocatalytic activity. Energy-dispersive X-ray spectroscopy confirmed the presence of only Zn and O elements, while scanning electron microscopy showed uniformly dispersed rod-like morphologies with particle sizes in the range of 10-25 nm. Powder X-ray diffraction confirmed a crystalline hexagonal wurtzite phase with an average crystallite size of 7.56 nm. The photocatalytic efficiency of ZnO nanorods was estimated using methylene blue, Congo red, and rhodamine B dyes as model pollutants, in addition to real textile wastewater samples. The treated wastes showed improved dissolved oxygen from 0.4 to 2.13 mg/L and lowered BOD from 930 to 30 mg/L. These data show the reproducibility and efficiency of green synthesis of ZnO nanoparticles using M. oleifera for wastewater purification.
AIM:The broad-spectrum antibacterial properties of silver nanoparticles (AgNPs) are attracting increasing attention as a means to combat drug-resistant infections. Herein, we describe the green production of AgNPs with minimal environmental impact by using plant components as reducing agents. METHODS AND RESULTS:Hibiscus rosa sinensis flower extract was used to form AgNPs from a 1 mM solution of silver nitrate. The NPs were cysteine-stabilized, on which hen egg white lysozyme was immobilized. The NPs, as analyzed by SEM, were nearly spherical. The size of the Cys-NPs, as measured by TEM, ranged from 12.52 nm to 27.20 nm, with an average size of 22.83, and the size of Lys-Cys-AgNPs ranged from 16.32 nm to 44.60 nm, with an average size of 30.30 nm. The enzyme assay for Lys-Cys-AgNPs demonstrated an effective activity of approximately 80.8 ± 3.6% compared to free lysozyme, and the immobilization efficiency was calculated to be 66.8%. MIC of Lys-Cys-AgNPs against E. coli (8.5 μg/mL), P. aeruginosa (12 μg/mL), S.aureus (6 μg/mL) and S. mutans (5.5 μg/mL). The antibacterial activity of Lys-Cys-AgNPs combined with streptomycin was evaluated against E. coli, P. aeruginosa, S. aureus, and S. mutans using the agar well diffusion method. The zones of inhibition observed were 11.00 ± 0.31 mm, 8.00 ± 0.11 mm, 8.00 ± 0.14 mm and 18.00 ± 0.65 mm, respectively. CONCLUSION:Green-synthesized, cysteine-stabilized silver nanoparticles functionalized with lysozyme demonstrated enhanced antibacterial efficacy against both Gram-positive and Gram-negative pathogens. These findings highlight their potential as an eco-friendly and effective nanobiotherapeutic strategy to combat drug-resistant infections.
This study optimized the extraction of polyphenols from Dendrobium residue using a natural deep eutectic solvent (NADES). A Box-Behnken design determined the optimal conditions: a choline chloride to glucose molar ratio of 2.4:1, solid-to-solvent ratio of 1:61 g/mL, water content of 35.7%, temperature of 80 °C, and duration of 120 minutes. This yielded a polyphenol content of 1.381 ± 0.071%, which was 2.93 to 38.36 times higher than conventional organic solvent methods. Fourier transform infrared (FTIR) spectroscopy was used to characterize the hydrogen bonding changes in the NADES system, while scanning electron microscopy (SEM) was employed to examine the morphological differences of Dendrobium residues before and after extraction treatment. Ten compounds were identified from the NADES extract, namely Prunin, Schaftoside, Apigenin-6,8-di-C-α-l-arabinopyranoside, Batatasin III, Moscatilin, Dihydroresveratrol, Hesperetin, Naringenin, Dendrophenol, and Erianin, which exhibited important pharmacological activities. Ultra-performance liquid chromatography(UPLC) analysis revealed a great variety and quantity of polyphenols in the NADES extract. Furthermore, the extract exhibited superior antioxidant activity: at 1.00 mg/mL, DPPH scavenging was 90.615 ± 0.92%, ABTS scavenging was 96.095 ± 0.44%, and FRAP value was 1.048 ± 0.03 mmol/L. These results highlight the green and efficient nature of this NADES-based protocol for industrial applications in food and herb residue processing.
