Search Results (233)

The use of hydrolytic enzymes is one of the most promising methods for combating bacterial biofilms. However, the use of native enzymes is limited by the rapid loss of activity under unfavorable conditions. Immobilization of enzymes on carbon nanoparticles enhances their stability, allows for biocatalyst reuse, and creates a synergistic effect due to the intrinsic antimicrobial properties of the nanomaterials. The aim of this investigation was to create and comparatively analyze conjugates of acid and alkaline proteases with carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) and to assess their effect on the formation and destruction of E. coli VKM B-3858D biofilms. The immobilization efficiency and kinetics of enzyme adsorption on the support were quantified by determining the protein concentration using the Bradford assay. The morphology and dispersion of the resulting conjugates were analyzed using atomic force microscopy (AFM). Protease activity was determined by a modified Anson method using the Folin–Ciocalteu reagent. Biofilm biomass was determined using crystal violet staining. The binding efficiency of the acid protease to MWCNTs-COOH was shown to reach 93%, which is significantly higher than that of the alkaline protease. The highest degree of immobilization was observed at a protein concentration of 117–338 μg/mL (10–20 mg/mL of the enzyme preparations). The interaction of the acid protease with the carbon nanoparticles increased dispersion, reducing the size of aggregates from ~1 μm to ~68 nm. As a result, acid protease conjugates with MWCNTs-COOH significantly reduced the biofilm biomass compared to both the enzyme-free control and the native enzyme. Alkaline protease, unlike the acid protease, destroys mature biofilms, and immobilization on MWCNTs-COOH enhances this ability. Native alkaline protease and acid protease conjugates with MWCNTs-COOH are effective in combating the biofilm formation of Gram-negative bacteria, while alkaline protease conjugates are suitable for disrupting mature biofilms. Full article

Plant growth-promoting fungi (PGPF) have shown broad potential to improve soil conditions and enhance root growth and development. However, few studies have examined the effects of exogenous PGPF inoculation on the growth of the medicinal plant Saposhnikovia divaricata and the associated changes in the rhizosphere microbiome. In this study, Aspergillus neoalliaceus MR-86 exhibited phosphate solubilization, growth in nitrogen-free medium, potassium solubilization, IAA production, and siderophore production. PCR assays did not detect the aflatoxin biosynthesis-related genes aflR, aflS, and omtA in strain MR-86. Pot trials demonstrated that inoculation with MR-86 significantly increased the plant height and root dry weight of S. divaricata by 10.32% and 21.05%, respectively (p < 0.05). In the rhizosphere, soil pH decreased, whereas soil alkaline-hydrolyzable nitrogen and available phosphorus levels, as well as the activities of protease, urease, and cellulase, increased significantly. Illumina NovaSeq sequencing revealed that MR-86 inoculation altered the soil microbial community structure and specifically enriched several microbial taxa, including Talaromyces, Subulicystidium, and Aspergillus. Moreover, MR-86 inoculation did not alter the composition of dominant bacterial and fungal phyla, but significantly modified microbial interactions and the topology of microbial networks. Correlation analysis indicated that the specific microbial taxa Subulicystidium, Aspergillus, and Talaromyces were positively associated with soil nutrient indices, enzyme activities, and plant growth parameters. Functional prediction analysis indicated that MR-86 treatment was predicted to be enriched bacterial metabolic pathways, including flavone and flavonol biosynthesis and ether lipid metabolism, and was predicted to increase the relative abundance of functional fungal groups such as ectomycorrhizal and wood-decomposing fungi. In summary, A. neoalliaceus MR-86 may contribute to improved growth of S. divaricata by enhancing nutrient availability and transformation and by modulating the structure and function of the rhizosphere microbiome. Full article

Tomato processing generates large amounts of by-products, with seeds representing an underutilized yet protein-rich fraction. This study investigated direct enzyme-assisted protein extraction from non-defatted tomato seeds. Various enzymes, enzyme/substrate ratios, pre-treatments, and incubation temperatures were evaluated and optimized. An enzyme/substrate ratio of 5% (w/w) was found to be optimal, with proteases alone outperforming cell wall-degrading enzymes and two-step extraction strategies. Bromelain, Protamex, and Trypsin, for the first time applied directly to non-defatted tomato seeds, achieved the highest protein recoveries (average 110.56 mg BSA eq/g DW). Among them, Trypsin also produced the highest reducing sugar content (25.07 mg GLU eq/g DW), indicating effective cell wall disruption. Digestates obtained from defatted and non-defatted tomato seeds showed comparable protein contents, demonstrating that defatting was unnecessary. Avoiding the defatting step improved process sustainability by reducing solvent use and energy consumption without significantly affecting protein extraction efficiency. Incubation at 37 °C was preferred over 60 °C, as similar yields were achieved under milder conditions while also reducing energy consumption by approximately three-fold (54,340 kJ vs 150,480 kJ for a 1000 L water-based scale-up simulation). These digestates showed significantly higher antioxidant and, for the first time in tomato seed extracts, anti-tyrosinase activities compared with controls. Protamex-derived samples exhibited the highest bioactivities (7.40 mg AA eq/g DW; 101.36 μg KA eq/g DW). Compared to conventional alkaline–acid extraction followed by enzymatic digestion, the direct enzymatic approach provided higher protein recovery. Overall, this method represents a sustainable strategy for producing bioactive peptide-rich extracts for food and non-food applications. Full article

This study was conducted to optimize the conditions for the synergistic treatment of cottonseed protein with microorganisms and enzymes and to evaluate the nutritional value and antioxidant activity of the resulting cottonseed peptides, with the ultimate goal of improving the nutritional quality of cottonseed protein. In single-factor experiments, laccase, alkaline protease, Saccharomyces cerevisiae, and Lactobacillus acidophilus were individually applied to cottonseed protein, and the optimal ranges for additive dosage, temperature, moisture content, and treatment duration were established using free gossypol, acid-soluble protein, and pH as response indicators. A Box–Behnken response surface design was subsequently adopted to perform an integrated analysis of the three responses and to determine the optimal conditions for the combined microbial–enzymatic treatment. The nutritional value and antioxidant activity of the cottonseed peptides obtained under these conditions were then evaluated. The optimal process parameters were identified as follows: microbial and enzyme dosages each at 1% (w/w), temperature of 37 °C, 37% moisture content, and treatment time of 96 h. Under the optimized conditions, the free gossypol content of the treated cottonseed protein was reduced to 67.30 mg/kg, representing a decrease of 83.69%; the acid-soluble protein content reached 29.72%, an increase of 25.86 percentage points; the reducing sugar content was 19.49 mg/g, an increase of 13.89 mg/g; and the pH dropped by 1.59 units to 4.91. Analysis of the peptide molecular weight distribution revealed that 99.61% of the cottonseed peptides had a molecular weight below 10,000 Da, and 65.45% were below 1000 Da. The peptides also exhibited excellent antioxidant capacity. In conclusion, the microbial–enzymatic synergistic treatment significantly elevated the contents of acid-soluble protein, reducing sugars, and peptides, enhanced antioxidant capacity, and reduced both free gossypol content and pH, thereby effectively improving the nutritional quality of cottonseed protein. Full article

The industrial extraction of chitin from shrimp shell waste conventionally employs corrosive chemical treatments, which pose significant environmental hazards and compromise polymer integrity. This study introduces a sustainable and highly efficient microbial biorefining strategy for the recovery of α-chitin from Litopenaeus vannamei shells, utilizing a sequential fermentation framework. Two potent strains—Bacillus haynesii MGPUMGRI, known for its proteolytic capabilities, and Lactobacillus delbrueckii MGPUMGRI, which produces lactic acid—were isolated and optimized. A notable technical achievement was the purification of an approximately 40 kDa extracellular alkaline protease from B. haynesii, which demonstrated optimal activity at pH 9.0 and 37 °C. Under optimized conditions, the sequential process—emphasizing enzymatic deproteinization (72.30 ± 1.56%) followed by lactic acid-mediated demineralization (84.98 ± 1.96%)—achieved a high-purity chitin recovery of 61.33 ± 1.06%. Comprehensive characterization using SEM-EDX, FTIR, and XRD confirmed the successful preservation of the α-chitin polymorphic structure, which exhibited a fragmented fibrillar morphology and a crystallinity index (CrI) of 60.51%. These findings indicate that this dual-strain bioprocess offers a scalable and environmentally friendly alternative for the valorization of seafood waste into high-quality biogenic polymers, while minimizing the ecological impact of chitin production. Full article

In our preliminary work, a clam sauce prepared by fermentation with Aspergillus oryzae 3.042 (AO) exhibited desirable flavor and quality; however, the process was prolonged (exceeding 30 d), and a high salt concentration (6–15%) was necessary to prevent spoilage. Consequently, shortening production cycle and reducing salt content without compromising product quality became a new objective. Enzymatic hydrolysis has long been recognized as an efficient approach in seasoning production, with enzyme efficacy being a key competitive factor. Accordingly, an AO-derived aminopeptidase–protease complex (AOAP) was optimized and prepared as a preparatory step. In this study, AOAP was applied to hydrolyze clam meat to evaluate its potential for producing a seasoning base. A two-step enzymatic hydrolysis process was employed. In the first step, the highest hydrolysis degree (29.1%) was achieved using alkaline protease (AP). The resulting hydrolysate was subsequently subjected to secondary hydrolysis with AOAP, achieving a degree of hydrolysis as high as 49.8%. Sensory evaluation revealed a significant reduction in bitterness and enhancement of umami in the final hydrolysate, a finding corroborated by electronic tongue analysis. Further characterization via LC-MS and amino acid (aa) analysis showed that a substantial number of bitter and umami peptides were released following AP treatment; however, the number of these peptides was markedly reduced after a subsequent AOAP hydrolysis, with concurrent substantial changes in the peptide profile. In the two-step hydrolysate, umami peptides mostly contain 3–10 aa, whereas bitter peptides typically contain only 3–5 aa. The content of free aa increased from 369.17 mg/100 g in the control to 3026.25 mg/100 g in the two-step hydrolysate, half of which were bitter, indicating the debittering efficiency of AOAP. Electronic nose analysis revealed similar flavor profile and characteristic presence of nitrogen oxides in all hydrolysates. GC-MS analysis further demonstrated that, after combined enzymatic hydrolysis, the short-chain aldehydes and ketones responsible for the fishy odor in the raw material almost completely disappeared, while long-chain aldehydes with pleasant aromas were generated. These findings suggest that the secondary hydrolysis step using AOAP can effectively improve the overall flavor profile of the clam hydrolysate, which may support its potential applicability in seasoning production, though further optimization and scale-up validation are needed. Full article

To improve the flavor quality and antioxidant activity of walnut gluten peptides, gluten was extracted from defatted walnut meal by alkaline solubilization and acid precipitation, hydrolyzed with alkaline protease to prepare antioxidant peptides, and further modified by the Maillard reaction. The optimal sugar source was selected by single-factor experiments, and reaction conditions were optimized by response surface methodology. Peptide conformational changes were characterized by UV, fluorescence, DSC, FTIR, and SEM, while changes in amino acid composition, flavor properties, and antioxidant activity were systematically evaluated. Fructose was identified as the optimal sugar source. The optimal reaction conditions were a peptide-to-sugar ratio of 1:1.2, 78.5 °C, initial pH 7.6, and 2 h reaction time, under which the sensory score reached 8.5 and DPPH radical scavenging activity reached 66.92%. Maillard modification markedly altered peptide conformation, as shown by increased UV absorbance, decreased intrinsic fluorescence intensity with a red shift, an increase in denaturation temperature from 80 °C to 100 °C, reduced α-helix content, increased β-sheet content, and transformation of the microstructure from a loose porous morphology to dense block-like aggregates. Free amino acid content increased initially and then decreased, whereas total essential amino acids were largely retained, indicating that the overall nutritional composition was preserved. However, further evaluation of digestibility and bioavailability is required to confirm nutritional value. These findings provide a feasible strategy for improving the flavor and functional properties of walnut gluten peptides and support their high-value utilization. Full article

The estuarine and coastal regions of India and Taiwan are under increasing threat from pollutants such as microplastics (MPs) and heavy metals including cadmium (Cd). These contaminants are known to have adversely affect biodiversity and water quality. In this study, the combined toxic effects of polyethylene microplastics (PE-MPs) and Cd were evaluated using Poecilia sphenops, a euryhaline fish species, selected for its adaptability to varying salinity conditions. P. sphenops were exposed to Cd (20, 40, and 60 μg/L), MPs (8, 16, 24 mg/L), and co-exposure combinations ranging from Cd 5 μg/L + MPs 4 mg/L to Cd 20 μg/L + MPs 16 mg/L Results showed significant (p < 0.05) negative effects on growth parameters including body weight gain, specific growth rate (SGR), and survival rate. Hematological analysis revealed significant (p < 0.05) decreases in hemoglobin (Hb), red blood cells (RBCs), and white blood cells (WBCs), indicating impaired oxygen transport and compromised immune function. Elevated blood glucose levels indicated physiological stress, while reduced total protein levels suggested a compromised nutritional status. Antioxidant enzyme activities, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), were significantly (p < 0.05) decreased in the toxicant-treated groups compared with the control. Digestive enzyme activities (proteases, amylases, and lipases) were also reduced, suggesting impaired digestion and nutrient assimilation. The study also included a comparative assessment of water quality between the exposed and control tanks. Water quality parameters such as turbidity, salinity, hardness, alkalinity, chloride, fluoride, and total suspended solids (TSSs) were elevated in the toxicant-treated media, accompanied by a notable decline in dissolved oxygen (DO) levels. These findings highlight the urgent need for integrated pollution control and water quality monitoring, particularly in coastal regions vulnerable to desalination discharges and plastic contamination. Sustainable management strategies must address these complex interactions between multiple pollutants to protect aquatic ecosystems. Full article

Proteases are key biocatalysts widely applied in the food, pharmaceutical, detergent, and environmental industries. One of the most costly steps in large-scale enzyme production is the preparation of the culture medium, making agro-industrial wastes attractive as low-cost nutrient sources and potential inducers. The non-conventional yeast Yarrowia lipolytica stands out in bioprocess engineering due to its high secretion capacity, GRAS status, and ability to metabolize diverse industrial residues. In this study, Brazilian agro-industrial by-products, namely Corn steep liquor (CSL), brewer’s yeast residue (BYR), and okara, were evaluated as alternative nitrogen sources for protease production by Y. lipolytica IMUFRJ 50678. Enzyme activity was quantified by the azocasein method at optimized conditions (40 °C, 40 min, pH 5 and 8). After an initial exploratory screening (n = 1), brewer’s yeast residue (BYR) and okara were identified as promising candidates for protease production. These preliminary findings guided subsequent experiments performed in biological triplicate (n = 3), which confirmed the reproducibility and comparative performance of these substrates, showing higher acid protease (AXP) activity in the BYR medium ((5.4 ± 0.3) U/mL), whereas alkaline protease (AEP) activities were comparable between the BYR ((8.4 ± 0.6) U/mL) and okara ((7.5 ± 0.9) U/mL) media. CSL was associated with higher lipase activity ((11.7 ± 0.9) × 10³ U/L), while esterase activity was higher in the BYR medium. These findings indicate that agro-industrial residues, particularly BYR and okara, can serve as effective nitrogen sources for protease production by Y. lipolytica IMUFRJ 50678, supporting their use in waste valorization and sustainable bioprocesses. Full article

Native Staphylococcus aureus protein A exhibits strong affinity to the Fc and VH regions of human IgG1, IgG2, and IgG4, making it a valuable tool for monoclonal antibody (mAb) purification. However, its low stability under conditions such as increased alkaline concentrations during cleaning-in-place (CIP), protease exposure, thermal stress, and shear forces limits its usability for large-scale industrial applications. Recombinant Protein A (rProtein A) can be modified to improve key properties, including alkaline stability. In this study, we present targeted modifications to the C domain of native Protein A, evaluating multimeric variants for structural and functional improvements. The selected variant demonstrated extremely high stability after 60 h incubation at 0.5 M NaOH by maintaining more than >90% initial dynamic binding capacity (DBC) and up to 80% DBC after 40 h in 1.0 M NaOH. However, the most impressive result obtained was the stability of the ligand in 1.5 M NaOH, retaining 80% DBC after 22 h and 60% DBC after 40 h. To the best of our knowledge, this is the first time that such high alkaline stability is reported for a rProtein A. To assess its application in monoclonal antibody purification, the optimized rProtein A ligand was immobilized on agarose resin and tested in chromatography processes. The resulting chromatography resin functionalized with the CmZmb ligand (now commercialized by Sunresin, China under the name of rProtein A Seplife Suno) exhibited a high dynamic binding capacity of 70 mg/mL, minimal ligand leaching under operational conditions (~15 ppm), and extended lifecycle performance (88% DBC retained after 120 purification cycles with 0.5 M NaOH CIP), making it well-suited for industrial-scale applications. Full article

Vibrio alginolyticus is a foodborne pathogen commonly found in seafood and freshwater products, causing human illness through the consumption of tainted seafood. Small non-coding RNAs (sRNAs) take effect on the stability and translation of their target mRNAs by base-pairing, thereby quickly altering bacterial physiology and pathogenicity at the post-transcriptional level. This work constructed a label-free in-frame deletion mutant and a complement strain of micX, a cell-density-associated sRNA in V. alginolyticus. The ΔmicX mutant exhibited reduced growth and a reduction in the synthesis of exopolysaccharides, biofilm, and alkaline serine protease. A TMT-based quantitative proteomic analysis comparing ΔmicX with the wild-type strain identified 900 differentially expressed proteins, comprising 376 that were upregulated and 524 that were downregulated. The upregulated proteins are primarily associated with porin activity, transmembrane signaling receptor function, and the two-component system. The downregulated proteins are mainly engaged in processes including biofilm formation, cellular communication, and transmembrane transport activity. Of note, the expression levels of proteins involved in the type VI secretion system, exopolysaccharide synthesis, mannose-sensitive hemagglutinin type IV pili (MSHA), and biofilm formation were significantly reduced in the absence of micX. Furthermore, the expression levels of proteins associated with quorum sensing (particularly LuxR and AphA) changed significantly in the ΔmicX vs. WT comparison. These findings strengthened comprehension of the novel sRNA regulatory network and established a theoretical foundation for additional investigations into the virulence of V. alginolyticus. Full article

This study compared the effectiveness of alkaline protease, neutral protease, trypsin, and papain in hydrolyzing oyster proteins and evaluated the antioxidant activities of the resulting hydrolysates. Alkaline protease achieved the highest degree of hydrolysis (30.96%) and the highest proportion of peptides ≤1 kDa (64.23%). Papain showed the lowest hydrolysis degree (18.29%). After separation by Sephadex G-15 gel filtration chromatography, the resulting low-molecular-weight peptide fractions (≤1 kDa) from each hydrolysate exhibited higher in vitro antioxidant activity than the higher-molecular-weight fractions (>1 kDa). Notably, trypsin and papain-derived low-molecular-weight fractions (OPP-T2 and OPP-P2) demonstrated stronger DPPH radical scavenging and inhibition of linoleic acid autoxidation than those from alkaline and neutral proteases. Cell experiments revealed that all low-molecular-weight fractions effectively alleviated H₂O₂-induced oxidative damage in LO2 cells. OPP-T2 and OPP-P2 exhibited significantly stronger protection of cell membrane integrity and enhancement of superoxide dismutase (SOD) activity than OPP-A2 and OPP-N2 (p < 0.05). OPP-T2 also showed the most pronounced increase in glutathione peroxidase (GSH-Px) activity (p < 0.05). These findings demonstrate that protease selection critically influences hydrolysis efficiency and antioxidant activity, with molecular weight being a key determinant of peptide antioxidant capacity. This work provides a reference for the development and application of oyster peptides. Full article

Objectives: While several physiological functions of milt peptides have been discovered, the structural characteristics of Theragra chalcogramma milt peptides (TMP) and their anti-fatigue mechanisms remain unclear. Methods: TMP was obtained by hydrolysis via flavor enzyme and alkaline protease, and its structural characteristics were analyzed. A mice model of exercise-induced fatigue was established. The anti-fatigue effect of TMP was evaluated by determining the main biochemical indices in the serum, liver, and skeletal muscle of mice. Additionally, qPCR analysis was conducted to investigate its regulatory effects on relevant energy metabolism pathways. Results: TMP contained 18.2% branched-chain amino acids, with those with molecular weights below 1000 Da accounting for 91.6%. A total of 154 characteristic peptides, such as VPFPR and LPPGR, were identified from TMP, among which 64% of the peptides contained glutamic acid, arginine, or aspartic acid. Molecular docking of potential bioactive peptides to AMP-activated protein kinase (AMPK) revealed binding energies from −9.1 to −5.5 kcal/mol. The exhaustive swimming test showed that oral administration of TMP prolonged the swimming duration. In the fatigue murine model, TMP reduced blood urea nitrogen and blood lactic acid levels while enhancing the content of muscle glycogen. Meanwhile, TMP significantly increased the activity of glutathione peroxidase and superoxide dismutase and reduced the accumulation of malondialdehyde, demonstrating antioxidant properties. Additionally, TMP significantly decreased creatine kinase and lactate dehydrogenase extravasation, thereby protecting muscle tissue, as corroborated by immunohistochemical analyses. Mechanistically, TMP upregulated AMPK and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) expression, promoting mitochondrial biogenesis via the AMPK/PGC-1α pathway. Conclusions: These findings suggest TMP has potential as a dietary supplement for alleviating physical fatigue. Full article

This study investigated the α-glucosidase inhibitory potential of enzymatic/alkaline treatments from Palmaria palmata using different proteases and pairwise combinations thereof. Treatments prepared with Alcalase^®, Flavourzyme^®, and Formea^® Prime, alone or in combination, were evaluated for dose-dependent inhibitory activity. Alcalase^®-derived treatments exhibited the highest α-glucosidase inhibition, achieving an IC₅₀ of 2.48 mg·mL⁻¹, outperforming other treatments and combinations. Membrane fractionation of the Alcalase^®-derived treatment into >5 kDa, 3–5 kDa, 1–3 kDa, and <1 kDa fractions revealed a size-dependent trend, with the <1 kDa fraction showing the strongest inhibition (IC₅₀ of 1.94 mg·mL⁻¹). Three peptides, RADIPFRRA, DGIAEAWLG, and FWSQIFGVAF, from the <1 kDa fraction were identified as potential α-glucosidase inhibitors using the BIOPEP-UWM database and were further selected based on a Peptide Ranker score above 0.6 for in silico docking analyses. Docking revealed distinct binding modes: RADIPFRRA and DGIAEAWLG occupied the catalytic cleft, interacting with key residues (Asp518, Asp616, Trp481, Trp613) consistent with competitive inhibition, whereas FWSQIFGVAF bound to a peripheral site, suggesting potential allosteric modulation. Physicochemical analysis further highlighted differences in charge and isoelectric point correlating with their binding behavior. Together, these findings demonstrate that low-molecular-weight peptides derived from P. palmata proteins, particularly those generated by Alcalase^®, possess significant α-glucosidase inhibitory activity, and provide structural insights for the rational design of peptide-based modulators of carbohydrate metabolism. Full article

Quinoa (Chenopodium quinoa) is a promising source of plant proteins with the potential to produce bioactive peptides through enzymatic hydrolysis. This study aimed to extract quinoa protein and produce bioactive peptides using two microbial proteases: Alcalase (from Bacillus licheniformis) and Flavourzyme (from Aspergillus oryzae). The protein was extracted through alkaline solubilization and isoelectric precipitation, achieving a 72% yield. Hydrolysis was conducted for 4 h, and enzymatic activity was measured using the TNBS method to determine the degree of hydrolysis, while SDS-PAGE was used to analyze protein breakdown. The reaction was performed at controlled pH and temperature (Alcalase: 9.5 and 55 °C; Flavourzyme: 7 and 37 °C). Both enzymes achieved maximum hydrolysis at 60 min. Consequently, the separation and inhibitory capacity of angiotensin-converting enzyme (ACE-I) were tested at the first four time points (0, 20, 40, and 60 min). A wider variety and higher concentration of peptides smaller than 2 kDa were found in hydrolysates treated with Flavourzyme, which is associated with antihypertensive activity. The ACE-I assay showed greater activity at the end of hydrolysis. Inhibition percentages of 87.5 ± 2.11 were observed in hydrolysates with Flavourzyme, and 94.1 ± 1.11 in those with Alcalase. These findings indicate that quinoa protein, hydrolyzed with microbial proteases, is a feasible source of peptides with potential antihypertensive effects for use in functional foods and nutraceuticals. Full article