Abstract：
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 IC50 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 IC50 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.
摘要：
对鱼加工废弃物进行估价,以获得胶原蛋白和生物活性肽等增值产品是提高经济价值的重要策略,减少处置问题，并防止对环境和健康的有害影响。本研究旨在从台湾罗非鱼皮中分离出两种胶原蛋白肽，并制备包括纳米乳液（NE）在内的12种纳米肽，纳米脂质体(NL)，和不含叶酸/壳聚糖（FA/CH）或FA配体缀合的纳米金（NG），用于比较它们对肺癌细胞A549和正常肺细胞MRC5的抑制效率。酸溶性胶原蛋白（产量，21.58％）用0.5M乙酸提取，水解得到两种罗非鱼皮胶原蛋白肽TSCP1（482Da）和TSCP2（172Da），分别使用2.5％和12.5％的alcalase，样品与水的比例为1:30(w/v)，pH8，温度50°C，和水解时间6小时。胶原蛋白肽的表征揭示了1型胶原蛋白的存在与大量的氨基酸，包括甘氨酸（32.6-33.1％），丙氨酸（13.6-14.0%），脯氨酸（10.0-10.5%），和羟脯氨酸（7.3-7.6％）。TSCP1，TSCP2和12纳米肽对A549细胞的细胞毒性高于MRC5细胞，与TSCP1及其6个纳米肽相比,TSCP2及其6个纳米肽表现出更低的IC50。TSCP2-NE的平均粒径分别为15.7、33.6和16.0nm，TSCP2-NL,和TSCP2-NG，但在配体缀合后改变为14.4、36.3和17.9nm，TSCP2-NE-FA/CH和TSCP2-NL-FA/CH的ζ电位从负向正变化。所有纳米肽在抑制A549细胞的生长方面都比肽更有效，TSCP2-NL-FA/CH的IC50值最低（5.32μg/mL），其次是TSCP2-NE-FA/CH（8.3μg/mL），TSCP2-NE（22.4μg/mL），TSCP2-NL（82.7μg/mL），TSCP2-NG-FA（159.8μg/mL），TSCP2-NG(234.0μg/mL)和TSCP2(359.7μg/mL)。子G1，S，G2/M期剂量依赖性增加，细胞周期可能停滞在G2/M期。TSCP2、TSCP2-NE的坏死细胞比例最高,TSCP2-NE-FA/CH,和TSCP2-NL，而晚期凋亡细胞以TSCP2-NL-FA/CH为主，TSCP2-NG,和TSCP2-NG-FA。同样，TSCP2及其6个纳米肽显示了caspase-3，caspase-8和caspase-9活性的剂量依赖性升高，以执行细胞凋亡，配体缀合的纳米肽是最有效的，其次是纳米肽和肽。这项研究的结果表明了台湾罗非鱼皮的有效策略，以获得具有抗癌活性的胶原蛋白肽及其纳米肽，并为将来的体内研究奠定了基础。
