School of Medicine, Huaqiao University, Quanzhou 362021, China.
Centre for Marine Bioproduct Development, College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia.
Mar Drugs. 2023 Feb 24;21(3):146. doi: 10.3390/md21030146.
has been identified as a sustainable source of rich proteins with diverse functionalities and bioactivities. After extracting C-phycocyanin (C-PC) and lipids in a biorefinery process, the spent biomass still contains a large proportion of proteins with potential for biopeptide production. In this study, the residue was digested using Papain, Alcalase, Trypsin, Protamex 1.6, and Alcalase 2.4 L at different time intervals. The resulting hydrolyzed product with the highest antioxidative activity, evaluated through their scavenging capability of hydroxyl radicals, superoxide anion, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), was selected for further fractionation and purification to isolate and identify biopeptides. Alcalase 2.4 L was found to produce the highest antioxidative hydrolysate product after four-hour hydrolysis. Fractionating this bioactive product using ultrafiltration obtained two fractions with different molecular weights (MW) and antioxidative activity. The low-molecular-weight fraction (LMWF) with MW <3 kDa had higher DPPH scavenging activity with the IC value of 2.97 ± 0.33 compared to 3.76 ± 0.15 mg/mL of the high-molecular-weight fraction (HMWF) with MW >3 kDa. Two stronger antioxidative fractions (F-A and F-B) with the respective significant lower IC values of 0.83 ± 0.22 and 1.52 ± 0.29 mg/mL were isolated from the LMWF using gel filtration with a Sephadex G-25 column. Based on LC-MS/MS analysis of the F-A, 230 peptides derived from 108 proteins were determined. Notably, different antioxidative peptides possessing various bioactivities, including antioxidation, were detected with high predicted scores together with in silico analyses on their stability and toxicity. This study established knowledge and technology to further value-add to the spent biomass by optimizing hydrolysis and fraction processes to produce antioxidative peptides with Alcalase 2.4 L after two products already produced in a biorefinery. These bioactive peptides have potential applications in food and nutraceutical products.
已将其鉴定为具有丰富功能和生物活性的可持续蛋白质来源。在生物炼制过程中提取藻蓝蛋白（C-PC）和脂质后，剩余的生物质仍含有很大比例的具有生物活性肽生产潜力的蛋白质。在这项研究中，使用木瓜蛋白酶、碱性蛋白酶、胰蛋白酶、Protamex 1.6 和碱性蛋白酶 2.4 L 在不同的时间间隔消化残留物。通过清除羟自由基、超氧阴离子、2,2-二苯基-1-苦基肼（DPPH）和 2,2'-联氨-双（3-乙基苯并噻唑啉-6-磺酸（ABTS）的清除能力来评估抗氧化活性，选择具有最高抗氧化活性的水解产物进行进一步的分级和纯化，以分离和鉴定生物活性肽。经过四小时水解，发现碱性蛋白酶 2.4 L 产生的抗氧化水解产物最高。使用超滤对这种生物活性产物进行分级，得到两种具有不同分子量（MW）和抗氧化活性的级分。MW<3 kDa 的低分子量级分（LMWF）的 DPPH 清除活性较高，IC 值为 2.97±0.33，而 MW>3 kDa 的高分子量级分（HMWF）的 IC 值为 3.76±0.15mg/mL。从 LMWF 中使用 Sephadex G-25 柱凝胶过滤分离出两种具有更强抗氧化性的级分（F-A 和 F-B），它们的 IC 值分别为 0.83±0.22 和 1.52±0.29mg/mL。基于 F-A 的 LC-MS/MS 分析，从 108 种蛋白质中确定了 230 种肽。值得注意的是，检测到了具有不同生物活性的不同抗氧化肽，包括抗氧化作用，同时还进行了稳定性和毒性的计算机分析，预测得分较高。这项研究建立了知识和技术，通过优化水解和分级过程，使用 Alcalase 2.4 L 从已在生物炼制中生产的两种产品后产生的剩余生物质中进一步生产具有抗氧化活性的肽，进一步增加剩余生物质的价值。这些生物活性肽在食品和营养保健品中有潜在的应用。
