Meng Lingting, Song Yan, Zheng Bin, Zhao Yadong, Hong Bingyuan, Ma Mingzhu, Wen Zhengshun, Miao Wenhua, Xu Yan
School of Food and Pharmacy, Zhejiang Ocean University, Zhejiang 316022, China.
School of Petrochemical Engineering & Environment, Zhejiang Ocean University, Zhejiang 316022, China.
Food Funct. 2023 Dec 11;14(24):10991-11004. doi: 10.1039/d3fo02948d.
To produce peptides with high dipeptidyl peptidase IV (DPP-IV) inhibitory activity, neutrase was selected from five proteases (trypsin, neutrase, pepsin, alcalase and flavor protease) with the highest degree of hydrolysis (DH) (18.23 ± 1.08%) and DPP-IV inhibitory rate (53.35 ± 4.02%) to produce protein hydrolysate (NPH) from the dark muscles of skipjack tuna (). Then, NPH-1 was isolated from NPH by gel permeation chromatography and found to possess the highest DPP-IV inhibitory rate (65.12 ± 7.94% at 0.5 mg ml) in the separated components (including NPH-1, NPH-2, NPH-3 and NPH-4). Subsequently, the available prediction models of tripeptides and tetrapeptides with the DPP-IV inhibitory rate were established using an artificial neural network (ANN). The RMSE (0.56 and 0.33 for the model established through collected tripeptides and tetrapeptides, respectively) and (0.95 and 0.99 for the model established through collected tripeptides and tetrapeptides, respectively) of the ANN model's parameters were within acceptable limits, indicating that this model is available. Next, the ANN model was applied to predict tripeptides and tetrapeptides from the hydrolysate of skipjack tuna dark muscles, and five peptides (Ala-Pro-Pro (APP), Pro-Pro-Pro (PPP), Asp-Pro-Leu-Leu (DPLL), Glu-Ala-Val-Pro (EAVP) and Glu-Ala-Iie-Pro (EAIP)) possessing a noticeable DPP-IV inhibitory rate (with DPP-IV IC values of 42.46 ± 5.02, 37.71 ± 9.17, 58.85 ± 14.42, 49.94 ± 6.69 and 57.15 ± 6.13 μM, respectively) were screened from the protein hydrolysate. The above five peptides were proved to effectively promote glucose consumption in the insulin resistant-HepG2 (IR-HepG2) cell model considering that the glucose consumption rates of APP, PPP, DPLL, EAVP and EAIP treatment groups are all more than twice that of the dexamethasone group. Accordingly, mechanistic studies showed that these peptides interacted with PI3K/AKT and AMPK signaling pathways and promoted the phosphorylation of PI3K p110, AKT and AMPK (the protein expressions of PI3K p110, p-AKT and p-AMPK in APP, PPP, DPLL, EAVP and EAIP treatment groups are 1.64-2.22 fold compared with that in the dexamethasone group), thereby enhancing glucose uptake and further alleviating insulin resistance. These findings demonstrated that skipjack tuna dark muscle is a potential DPP-IV inhibitory peptide source, and five DPP-IV inhibitory peptides from its hydrolysate may exert potent anti-diabetic activity. In comparison, PPP may be the most potential active ingredient for healthy food against type 2 diabetes mellitus in the five screened peptides considering synthetically the DPP-IV inhibitory rate, bioavailability and synthesis cost.
为了生产具有高双肽基肽酶IV（DPP-IV）抑制活性的肽，从五种蛋白酶（胰蛋白酶、中性蛋白酶、胃蛋白酶、碱性蛋白酶和风味蛋白酶）中选择了水解度（DH）最高（18.23±1.08%）和DPP-IV抑制率（53.35±4.02%）的中性蛋白酶，用于从鲣鱼的暗色肌肉中制备蛋白水解物（NPH）。然后，通过凝胶渗透色谱法从NPH中分离出NPH-1，发现在分离的组分（包括NPH-1、NPH-2、NPH-3和NPH-4）中，NPH-1具有最高的DPP-IV抑制率（在0.5mg/ml时为65.12±7.94%）。随后，使用人工神经网络（ANN）建立了具有DPP-IV抑制率的三肽和四肽的可用预测模型。ANN模型参数的均方根误差（通过收集的三肽和四肽建立的模型分别为0.56和0.33）和决定系数（通过收集的三肽和四肽建立的模型分别为0.95和0.99）在可接受范围内，表明该模型可用。接下来，将ANN模型应用于预测鲣鱼暗色肌肉水解物中的三肽和四肽，并从蛋白水解物中筛选出五种具有显著DPP-IV抑制率的肽（丙氨酸-脯氨酸-脯氨酸（APP）、脯氨酸-脯氨酸-脯氨酸（PPP）、天冬氨酸-脯氨酸-亮氨酸-亮氨酸（DPLL）、谷氨酸-丙氨酸-缬氨酸-脯氨酸（EAVP）和谷氨酸-丙氨酸-异亮氨酸-脯氨酸（EAIP））（其DPP-IV IC值分别为42.46±5.02、37.71±9.17、58.85±14.42、49.94±6.69和57.15±6.13μM）。考虑到APP、PPP、DPLL、EAVP和EAIP处理组的葡萄糖消耗率均是地塞米松组的两倍以上，上述五种肽被证明能有效促进胰岛素抵抗的HepG2（IR-HepG2）细胞模型中的葡萄糖消耗。因此，机制研究表明，这些肽与PI3K/AKT和AMPK信号通路相互作用，并促进PI3K p110、AKT和AMPK的磷酸化（APP、PPP、DPLL、EAVP和EAIP处理组中PI3K p110、p-AKT和p-AMPK的蛋白表达与地塞米松组相比为1.64-2.22倍），从而增强葡萄糖摄取并进一步减轻胰岛素抵抗。这些发现表明，鲣鱼暗色肌肉是一种潜在的DPP-IV抑制肽来源，其水解物中的五种DPP-IV抑制肽可能具有强大的抗糖尿病活性。相比之下，综合考虑DPP-IV抑制率、生物利用度和合成成本，PPP可能是五种筛选肽中对抗2型糖尿病的健康食品最具潜力 的活性成分。
