An Integrated Peptidomics and In Silico Approach to Identify Novel Anti-Diabetic Peptides in Parmigiano-Reggiano Cheese

SIMPLE SUMMARY: Dietary habits and sedentary lifestyle are widely established to be the major risk factors for the development of long-term chronic conditions, such as type-2 diabetes (T2D). In this framework, where consumer’s food choices are even more influenced by an ever-growing awareness on nutritional, environmental, and healthy aspects, the presence and identification of natural bioactive compounds are gaining increasing attention in the scientific community. Pharmacological treatment of T2D is based on the administration of molecules able to inhibit some key enzymes involved in the carbohydrate digestion and insulin secretion. The multiple side-effects of synthetic inhibitors led to an increased demand for natural food-derived anti-diabetic agents. The present work offers a new integrated approach for the identification and selection of new bioactive peptides, able to inhibit the key digestive enzymes implicated in the control of blood glucose level. The novelty lies in the development of a new, quick, and cost-effective integrated methodology supported by confirmed in vitro evidence. In fact, the present work successfully accomplished the identification of two selected candidates with a possible application in the diabetes management. Indeed, the functional and healthy role of Parmigiano-Reggiano cheese in human nutrition was assessed, highlighting its potential anti-diabetic properties. ABSTRACT: Inhibition of key metabolic enzymes linked to type-2-diabetes (T2D) by food-derived compounds is a preventive emerging strategy in the management of T2D. Here, the impact of Parmigiano-Reggiano (PR) cheese peptide fractions, at four different ripening times (12, 18, 24, and 30 months), on the enzymatic activity of α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPP-IV) as well as on the formation of fluorescent advanced glycation end-products (fAGEs) was assessed. The PR peptide fractions were able to inhibit the selected enzymes and fAGEs formation. The 12-month-ripening PR sample was the most active against the three enzymes and fAGEs. Mass spectrometry analysis enabled the identification of 415 unique peptides, 54.9% of them common to the four PR samples. Forty-nine previously identified bioactive peptides were found, mostly characterized as angiotensin-converting enzyme-inhibitors. The application of an integrated approach that combined peptidomics, in silico analysis, and a structure–activity relationship led to an efficient selection of 6 peptides with potential DPP-IV and α-glucosidase inhibitory activities. Peptide APFPE was identified as a potent novel DPP-IV inhibitor (IC(50) = 49.5 ± 0.5 μmol/L). In addition, the well-known anti-hypertensive tripeptide, IPP, was the only one able to inhibit the three digestive enzymes, highlighting its possible new and pivotal role in diabetes management.