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Valorization of fish processing waste to obtain value-added products such as collagen and bioactive peptides is a vital strategy to increase the economic value, reduce disposal problems, and prevent harmful impacts on both environment and health. This study aims to isolate two collagen peptides from Taiwan Tilapia skin and prepare 12 nanopeptides including nanoemulsion (NE), nanoliposome (NL), and nanogold (NG) without and with folic acid/chitosan (FA/CH) or FA ligand conjugation for comparison of their inhibition efficiency towards lung cancer cells A549 and normal lung cells MRC5. Acid-soluble collagen (yield, 21.58 %) was extracted using 0.5 M acetic acid and hydrolyzed to obtain two tilapia skin collagen peptides TSCP1 (482 Da) and TSCP2 (172 Da) respectively using 2.5 % and 12.5 % alcalase, with sample-to-water ratio at 1:30 (w/v), pH 8, temperature 50 °C, and hydrolysis time 6 h. Characterization of collagen peptides revealed the presence of type 1 collagen with a high amount of amino acids including glycine (32.6-33.1 %), alanine (13.6-14.0 %), proline (10.0-10.5 %), and hydroxyproline (7.3-7.6 %). TSCP1, TSCP2, and 12 nanopeptides showed a higher cytotoxicity towards A549 cells than MRC5 cells, with TSCP2 and its 6 nanopeptides exhibiting a lower IC 50 compared to TSCP1 and its 6 nanopeptides. The mean particle size was 15.7, 33.6, and 16.0 nm respectively for TSCP2-NE, TSCP2-NL, and TSCP2-NG, but changed to 14.4, 36.3, and 17.9 nm following ligand conjugation with a shift in zeta potential from negative to positive for TSCP2-NE-FA/CH and TSCP2-NL-FA/CH. All nanopeptides were more effective than peptides in inhibiting the growth of A549 cells, with the lowest IC 50 value being shown for TSCP2-NL-FA/CH (5.32 μg/mL), followed by TSCP2-NE-FA/CH (8.3 μg/mL), TSCP2-NE (22.4 μg/mL), TSCP2-NL (82.7 μg/mL), TSCP2-NG-FA (159.8 μg/mL), TSCP2-NG (234.0 μg/mL) and TSCP2 (359.7 μg/mL). Cell proportions of sub-G1, S, and G2/M phases increased dose-dependently, with a possible cell cycle arrest at G2/M phase. The proportion of necrotic cells was the highest for TSCP2, TSCP2-NE, TSCP2-NE-FA/CH, and TSCP2-NL, while that of late apoptotic cells dominated for TSCP2-NL-FA/CH, TSCP2-NG, and TSCP2-NG-FA. Similarly, TSCP2 and its 6 nanopeptides showed a dose-dependent rise in caspase-3, caspase-8, and caspase-9 activities for execution of apoptosis, with the ligand-conjugated nanopeptides being the most efficient, followed by nanopeptides and peptides. The outcome of this study demonstrated an effective strategy for valorization of Taiwan tilapia skin to obtain collagen peptides and their nanopeptides possessing anticancer activity and form a basis for in vivo study in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Animal testing has been the primary approach to assess the neutralization potency of antivenom for decades. However, the necessity to sacrifice large numbers of experimental animals during this process has recently raised substantial welfare concerns. Furthermore, the laborious and expensive nature of animal testing highlights the critical need to develop alternative in vitro assays. Here, we developed an antibody-detection enzyme-linked immunosorbent assay (ELISA) technique as an alternative approach to evaluate the neutralization potency of hyperimmunized equine plasma against B. multicinctus, a medically important venomous snake in Taiwan. Firstly, five major protein components of B. multicinctus venom, specifically, α-BTX, β-BTX, γ-BTX, MTX, and NTL, were isolated. To rank their relative medical significance, a toxicity score system was utilized. Among the proteins tested, β-BTX presenting the highest score was regarded as the major toxic component. Subsequently, antibody-detection ELISA was established based on the five major proteins and used to evaluate 55 hyperimmunized equine plasma samples with known neutralization potency. ELISA based on β-BTX, the most lethal protein according to the toxicity score, exhibited the best sensitivity (75.6 %) and specificity (100 %) in discriminating between high-potency and low-potency plasma, supporting the hypothesis that highly toxic proteins offer better discriminatory power for potency evaluation. Additionally, a phospholipase A 2 (PLA 2 ) competition process was implemented to eliminate the antibodies targeting toxicologically irrelevant domains. This optimization greatly enhanced the performance of our assay, resulting in sensitivity of 97.6 % and specificity of 92.9 %. The newly developed antibody-detection ELISA presents a promising alternative to in vivo assays to determine the neutralization potency of antisera against B. multicinctus during the process of antivenom production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)