Department of Health, Animal Science and Food Safety, University of Milan, 20133 Italy.
Hugh Sinclair Unit of Human Nutrition, Department of Food and Nutritional Sciences, University of Reading, RG6 6AP United Kingdom; Institute for Cardiovascular and Metabolic Research, University of Reading, RG6 6AP United Kingdom.
J Dairy Sci. 2019 Dec;102(12):10760-10771. doi: 10.3168/jds.2019-16833. Epub 2019 Sep 11.
Over the past decades, several studies investigated the health-promoting functions of milk peptides. However, to date many hurdles still exist regarding the widespread use of milk-derived bioactive peptides, as they may be degraded during gastrointestinal digestion. Thus, the aim of our study was to in vitro digest intact whey protein isolate (WPI) and casein proteins (CNP), mimicking in vivo digestion, to investigate their bioactive effects and to identify the potential peptides involved. Whey protein isolate and CNP were digested using a pepsin-pancreatin protocol and ultra-filtered (3-kDa cutoff membrane). A permeate (<3 kDa) and a retentate (>3 kDa) were obtained. Soy protein was included as a control (CTR). Angiotensin-1-converting enzyme inhibitory (ACE1-I) and antioxidant activity (AOX) were assessed and compared with those observed in undigested proteins and CTR. Furthermore, the permeate was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and retentate was further digested with trypsin and analyzed by MS using a shotgun proteomic approach to identify potentially bioactive peptides. Further, the effects of WPI, CNP, and CTR retentate on cell metabolic activity and on mucus production (MUC5AC and MUC2 gene expression) were assessed in intestinal goblet HT29-MTX-E12 cells. Results showed that WPI permeate induced a significant ACE1-I inhibitory effect [49.2 ± 0.64% (SEM)] compared with undigested WPI, CNP permeate, and retentate or CTR permeate (10.40 ± 1.07%). A significant increase in AOX (1.58 ± 0.04 and 1.61 ± 0.02 µmol of trolox AOX equivalents per mg of protein, respectively) upon digestion was found in WPI. Potentially bioactive peptides associated with ACE1-I and antihypertensive effects were identified in WPI permeate and CNP retentate. At specific concentrations, WPI, CNP, and CTR retentate were able to stimulate metabolic activity in HT29-MTX-E12 cells. Expression of MUC5AC was increased by CNP retentate and unaltered by WPI retentate; MUC2 expression was significantly increased by 0.33 mg/g of CNP and reduced by 1.33 mg/g of CNP. Our results confirm that milk proteins may be rich sources of bioactive compounds, with the greatest beneficial potential of CNP at the intestinal goblet cell level.
在过去的几十年中，已有多项研究调查了牛奶肽的促进健康功能。然而，迄今为止，由于在胃肠道消化过程中可能发生降解，因此广泛使用牛奶来源的生物活性肽仍存在许多障碍。因此，我们的研究目的是通过体外消化完整的乳清蛋白分离物（WPI）和酪蛋白（CNP）来模拟体内消化，以研究它们的生物活性作用并鉴定潜在涉及的肽。使用胃蛋白酶-胰蛋白酶方案和超滤（3 kDa 截止膜）消化乳清蛋白分离物和酪蛋白。获得了透过物（<3 kDa）和保留物（>3 kDa）。将大豆蛋白作为对照（CTR）包含在内。评估了血管紧张素转化酶抑制（ACE1-I）和抗氧化活性（AOX），并将其与未消化的蛋白质和 CTR 观察到的进行了比较。此外，通过纳升液相色谱电喷雾串联质谱（LC-nano ESI MS/MS）使用 shotgun 肽组学方法对透过物进行了表征，并且用胰蛋白酶进一步消化保留物，并通过使用 shotgun 蛋白质组学方法进行 MS 分析来鉴定潜在的生物活性肽。此外，还评估了 WPI、CNP 和 CTR 保留物对肠道杯状 HT29-MTX-E12 细胞代谢活性和粘液产生（MUC5AC 和 MUC2 基因表达）的影响。结果表明，与未消化的 WPI、CNP 透过物、保留物或 CTR 透过物相比，WPI 透过物诱导 ACE1-I 抑制作用具有显着的效果[49.2±0.64%（SEM）]。在 WPI 消化过程中，发现 AOX 显着增加（分别为 1.58±0.04 和 1.61±0.02 µmol trolox AOX 当量/ mg 蛋白）。在 WPI 透过物和 CNP 保留物中鉴定出与 ACE1-I 和抗高血压作用相关的潜在生物活性肽。在特定浓度下，WPI、CNP 和 CTR 保留物能够刺激 HT29-MTX-E12 细胞的代谢活性。CNP 保留物增加了 MUC5AC 的表达，而 WPI 保留物则未改变；CNP 使 MUC2 的表达增加了 0.33mg/g，减少了 1.33mg/g。我们的研究结果证实，牛奶蛋白可能是生物活性化合物的丰富来源，CNP 在肠杯状细胞水平上具有最大的有益潜力。
