Recent evidence suggests that mulberry leaves have good antioxidant activity. However, what the antioxidant ingredient is and how the ingredient works are still not well understood. In this study, we enzymatically hydrolyze mulberry leaf proteins (MLPs) using neutral protease and find that the mulberry leaf protein hydrolysates (MLPHs) have stronger antioxidant activity compared to MLPs. We separate the core antioxidant components in MLPHs by ion-exchange columns and molecular sieves and identify 798 antioxidant peptides by LC-MS/MS. Through bioinformatics analysis and biochemical assays, we screen two previously unreported peptides, P6 and P7, with excellent antioxidant activities. P6 and P7 not only significantly reduce ROS in cells but also improve the activities of the antioxidant enzymes SOD and CAT. In addition, both peptides are found to exert protective effects against H₂O₂-induced chromatin damage and cell apoptosis. Collectively, these results provide support for the application of mulberry leaf peptides as antioxidants in the medical, food and livestock industries.

Oxidative stress and inflammatory responses often occur concomitantly, and they are key causative factors in various human and animal diseases. Evidence suggests that mulberry leaf protein (MLP) may have potential antioxidant and anti-inflammatory properties, but there are significant challenges in enhancing their bioactivities. In this study, MLP was enzymatically hydrolyzed using papain, protamex, alkaline protease, trypsin, and neutral protease, followed by comprehensive evaluation of the antioxidant capacity, anti-inflammatory properties, and cytotoxicity of the hydrolysates. Our findings revealed that some enzymes significantly enhanced the peptide production and antioxidant activity of MLP (p < 0.01), and its activity was positively correlated with the degree of hydrolysis. Among the five hydrolysates, neutral protease hydrolysate (NeuH) exhibited the best antioxidant properties, with free radical scavenging rates of 71.58 ± 0.42% (ABTS), 26.38 ± 0.15% (OH), and 73.91 ± 0.37% (DPPH) at a concentration of 0.1 mg/mL. In addition, NeuH significantly suppressed IL-6 secretion (p < 0.01) and downregulated mRNA expression of IL-6, iNOS, and COX-2 inflammatory markers. This study not only establishes a correlation between enzymatic parameters and MLP biological functions but also demonstrates the potential of optimized MLP hydrolysates, particularly NeuH, as valuable natural antioxidant and anti-inflammatory ingredients for functional foods or nutraceuticals aimed at mitigating oxidative stress and inflammation-related disorders.

With the increasing recognition of the role of immunomodulation and oxidative stress in various diseases, designing peptides with both immunomodulatory and antioxidant activities has emerged as a promising therapeutic strategy. In this study, a hybridization design was applied as a powerful method to obtain multifunctional peptides. A total of 40 peptides with potential immunomodulatory and antioxidant activities were designed and screened. First, molecular docking was employed to screen peptides with a high binding affinity to MD2, a key receptor protein in the NFκB immune pathway. For the in vitro high-throughput screening, we constructed a reporter gene-based stable cell line, IPEC-J2-Lucia ARE cells, which was subsequently used to screen peptides with antioxidant activity. Furthermore, the biocompatibility, immunomodulatory, and antioxidant activities of these peptides were assessed. Among the candidates, the hybrid peptide VA exhibited the strongest immune-enhancing activity through the activation of the NF-κB pathway and significant antioxidant activity via the Nrf2-ARE pathway. Additionally, VA demonstrated protective effects against H₂O₂-induced oxidative damage in HepG2 cells. This study not only demonstrates the potential of peptide hybridization, but also develops a screening platform for multifunctional peptides. It provides a new tool for the treatment of autoimmune diseases and oxidative stress-related diseases.

Oxidative stress plays a crucial role in the development and progression of various diseases. Antioxidant peptides have attracted great attention in agricultural, food, and clinical fields due to their low toxicity, high efficacy, and easy absorption, but the development of antioxidant peptides and their in-depth molecular mechanisms are still lacking. The previous study established a platform for the high-throughput design and screening of multifunctional peptides and successfully identified a novel hybrid peptide, VLP-Aβ (VA), which exhibits both antioxidant and immunomodulatory properties. This study aimed to evaluate the antioxidant activity of VA and investigate the underlying molecular mechanisms. The antioxidant effects of VA were evaluated using both in vitro (H₂O₂-induced oxidative damage in HepG2 cells) and in vivo (CCl₄-induced liver damage in mice) models. VA exhibited significant antioxidant activity both in vitro and in vivo, significantly improving the cell viability and increasing the levels of antioxidant enzymes (SOD, CAT, GSH-Px) to alleviate oxidative stress. These findings indicated that the antioxidant effect of VA is dependent on NRF2, as evidenced by NRF2 knockdown experiments. Further investigation revealed that VA alleviates oxidative stress by modulating the KEAP1-NRF2-ARE signaling pathway. These findings provide insights into the properties of the antioxidant peptide VA, expand the understanding of its molecular mechanisms, and suggest new opportunities for developing VA as a novel functional agent in the agricultural, food, and clinical industries.

Thymopentin (TP5) plays an important role in host immunomodulation, yet its bioavailability is significantly limited by its short half-life. YW12D is a peptide with strong stability but relatively weak immunoactivity. Tuning the physicochemical properties of such molecules may yield synthetic molecules displaying optimal stability, safety and enhanced immunological activity. Here, natural peptides were modified to improve their activity by hybridization strategies. A hybrid peptide YW12D-TP5 (YTP) that combines TP5 and YW12D is designed. The half-life of YTP in plasma is significantly longer than that of YW12D and TP5. YTP also displays an improved ability to protect the host from CTX-induced weight loss and thymus and spleen indices decrease than YW12D and TP5. In addition, YTP promotes dendritic cell maturation and increases the expression of cytokines IL-1β, IL-6, TNF-α and immunoglobulins IgA, IgG, and IgM. A combination of antibody-specific blocking assay, SPR, molecular dynamics simulations and western blotting suggest that the immunomodulatory effect of YTP is associated with its activation of the TLR2-NF-кB signaling axis. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

Microbially synthesized postbiotics have unique properties and advantages; however, systematic studies on the efficient production and biological functions of postbiotics from Bacillus subtilis are limited, which greatly restricts their applications. In this study, we obtained a novel crude exopolysaccharide (EPS) postbiotic from Bacillus subtilis H4. We systematically optimized the EPS production strategy using single-factor analysis, Plackett–Burman design, the path of steepest ascent method, and response surface methodology. The optimized EPS yield was significantly improved, with a maximum yield of 15.01 g/L under the addition of 4.12% soy peptone, 8.99% sucrose, and 0.06% MnSO₄. We found that EPS is a neutral, heterogeneous polysaccharide with a pyranose ring, with a molecular weight of 44,304.913 kDa and a melting point of 218 °C. It consists of glucose, galactose, arabinose, glucosamine, and mannose at a molar ratio of 58.85:19.81:14.75:10.89:6.58. EPS exhibits strong antioxidant capacities, scavenging ABTS and DPPH radicals with IC50 values of 1 and 6 mg/mL, respectively. Moreover, it shows notable anti-inflammatory properties, dramatically inhibiting the lipopolysaccharide (LPS)-induced elevation of nitric oxide (NO) levels and over-activation of the TLR4-NF-κB signaling pathway. These findings highlight the potential of EPS as a multifunctional bioactive compound, offering great promise for its application in the food, clinical, and feed industries.