Type II diabetes is a chronic disease, which affects millions of people worldwide: the inadequate secretion of insulin by β-cells and/or insulin resistance in tissues are its main characteristics (1). Dipeptidyl-peptidase IV (DPP-IV) is known for its inactivation of two intestinal hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which possess potent insulin-secretory activity, and consequently lower prandial plasma glucose (1). Due to the short half-life (< 2 min) of the degradation of GLP-1 and GIP by DPP-IV, the inhibition of DPP-IV is an attractive therapeutic strategy to maintain the insulinotropic activity of GLP-1 and GIP, resulting in an improved homeostasis of glucose in type II diabetes (2). Interestingly, a number of studies indicate that most people with diabetes will eventually develop hypertension and other blood circulation complications, especially showing a close association between Type II diabetes and hypertension (3, 4). There are many factors associated with the development of hypertension, one of them is related to the conversion of angiotensin I, an active decapeptide, into angiotensin II, a potent vasoconstrictor octapeptide, by the angiotensin-converting enzyme (ACE) system. Meanwhile, the inactivation of bradykinin, which shows a significant vasodilator activity, is caused by ACE. Therefore, inhibition of ACE is considered an effective treatment for lowering blood pressure in hypertensive subjects (5).

Inhibitors of DPP-IV and/or ACE are regularly applied in therapy to lower morbidity and mortality of patients with type II diabetes and/or hypertension. Synthetic inhibitors of DPP-IV and/or ACE are the first-line option for the treatment of these events (6), however, inevitable and evitable side effects of them, such as coughing, skin rashes, taste disturbances, angioedema, hyperkalaemia (ACE inhibitors) (7, 8), and nasopharyngitis, headache, diarrhea, joint pain, urinary tract infection (DPP-IV inhibitors) (9, 10), highlight the importance of developing new natural, innovative side-effect free compounds to prevent and/or alleviate diabetes and hypertension. Over the past few decades, many evidences have underlined that protein hydrolysates obtained from both plant (soybean, pea, rice bran, lupin, wheat, and many others) and/or animal (milk, egg yolk, beef, pork, chicken, sardine, salmon, and many others) foods may be useful for the prevention of these pathologies and several food derived peptides with potent DPP-IV-inhibitory and/or ACE-inhibitory activities have been successfully identified and characterized (11–16). These peptides are encrypted within the protein sequences and are released by the hydrolysis (17). They may be obtained with digestive enzymes (i.e., pepsin and trypsin), mimicking the action of in vivo gastrointestinal digestion, or produced using plant (i.e., bromelin) or microbial (i.e., Alcalase® and Flavourzyme®) food grade enzymes, as well as during some food manufacture processes, such fermentation (18, 19).

It has been clearly demonstrated (20) that milk protein derived peptides possess different biological properties, including antimicrobial (21), antioxidant (22), anti-inflammatory (23), antithrombotic, hypocholesterolemic (24) and anti-hypertensive (22) activities useful for cardiovascular risk prevention. Moreover, they are active in preventing nervous system and bone diseases (25, 26). Most of these bioactive peptides are generated from the degradation of casein, which makes up around 80% of the protein in cow milk and it is composed by α-, β- and κ-caseins (27).

In light with these considerations, this study had the goal of assessing the pleiotropic health-promoting effects of a commercial casein hydrolysate (CH), obtained by an industrial process. Since casein is an excellent source of biologically active peptides, it was hypothesized that this industrial product may possess a dual biological behavior, including DPP-IV-inhibitory and/or ACE-inhibitory activity. The first objective of the study was the characterization of the chemical composition of CH for identifying sequences that may be responsible for its biological activity. The second aim was the evaluation of the potential anti-diabetic and hypotensive effects of CH by measuring its inhibitory activities toward both DPP-IV and ACE. This was achieved by using either cell-free tests or cell assays based on human intestinal Caco-2 cells. Finally, the same experimental procedures were employed to evaluate the activities of the fraction with a molecular weight lower than 3 kDa [CH (F3)].

Undoubtedly, casein is among the most studied sources of bioactive peptides with health promoting activities. Indeed, several literature evidences report that casein acts as a precursor of a diversity of bioactive peptides with anti-hypertensive, immunological, antioxidant, antithrombotic and anti-diabetic effects (28–30). In this context, the ACE-inhibitory activity was the most investigated, and numerous ACE-inhibitory peptides have been identified, for instance, VPP (31), IPP (32), NMAINPSKENLCSTFCK (33), RYPSYG and DERF (34). In addition, also DPP-IV-inhibitory peptides have also been identified from casein and characterized, such as LPQNIPPL (35) and VPYPQ (36).

In this panorama, the main limitation of most previous studies on casein hydrolysate consists on the fact that the characterization of DPP-IV or ACE inhibitory activities has been carried out independently without taking into account that the same hydrolysate may be endowed with a multifunctional behavior exerting, therefore, both activities. Moreover, great part of published papers are based exclusively on cell-free assays (i.e., pure enzyme inhibition), whereas a main peculiarity of the present study is that the in vitro tests were implemented and substantiated by cellular assays that permitted either to get a deeper insight in the mechanism of action or to contextually consider other relevant issues, such as metabolism (this is particularly true while employing Caco-2 cells).

Overall, our findings suggested that CH is able to modulate the DPP-IV and ACE activities both in cell-free and cell-based conditions. More in details, CH displayed potent inhibitory properties against DPP-IV (IC₅₀ value of 0.38 ± 0.01 mg/mL) and ACE (IC₅₀ value of 0.39 ± 0.01 mg/mL) in cell-free condition, respectively. Furthermore, a comparison of the IC₅₀ values of complete hydrolysate with those of the low molecular weight fraction (<3 kDa) (Table 2), showed that medium and short-sized peptides, which account for about two thirds of the hydrolysate (62.05%), are mainly responsible of the observed biological activities.

Literature suggests that a camel total casein hydrolysate is less efficient than CH in reducing the ACE activity being its effect not observed up to 0.6 mg/mL. Whereas, the <3 kDa fraction of the same material displays an IC₅₀ equal to 91 ± 20 μg/mL (37). In addition, the IC₅₀ values of a thermolysin casein hydrolysate was 45–83 μg/mL (38). As regards the DPP-IV inhibitory activity, previous evidences suggest that casein hydrolysates showed IC₅₀ values in the range 0.88–1.1 mg/mL (39), suggesting that CH is slightly more active. In addition, as reported by Zhang et al., using different proteases, casein proteins released peptides that determined the 50% of DPP-IV inhibition at 1.25 μg/mL, clearly suggesting that CH is more active (40). Notably, recent evidences suggest that both soybean hydrolysate and pea hydrolysate inhibit DPP-IV and ACE activity with a dose–response trend and IC₅₀ values equal to 1.15 ± 0.004 and 1.33 ± 0.004 mg/mL, and 0.33 ± 0.01 and 0.61 ± 0.05 mg/mL, respectively (11).

In principle, the bioactivity of a food derived hydrolysate depends strictly on its total composition, including inactive and active species and possible synergistic or antagonist effects (41, 42). In light with this observation, Table 1 reveals that all the major identified peptides derived from CH included at least one motif with established DPP-IV and ACE inhibitory activity, which clarifies the reason why this peptide mixture is effective against both targets. Being the composition of casein hydrolysate naturally heterogeneous, it is feasible to consider that some peptides may be active against one of the two target, but we cannot exclude that some specific peptides may be contemporary endowed by the dual ability to target both ACE and DPP-IV. In addition, some peptides with post-translational modifications were also identified. Notably, it was observed that peptide VPNSAEER displays a phosphorylation on serine residue in position 4, whereas 6 different peptides (IPNPIGSENSEKTTMP, KEPMIGVN, SDIPNPIGSENSEKTTMP, EMPFPKYP, EMPFPKYPVEP, and MPFPKYPVEP) contain within their sequences oxidized methionine residue.

These results are in line with a previous work which focused on two commercially available soybean (SH) and pea (PH) protein hydrolysates (11). In particular, as regards the ACE inhibitory activity, CH displayed comparable IC₅₀ values with those obtained of both SH and PH, whereas as regards DPP-IV activity, CH hydrolysates is about 3.5-fold more potent than both SH and PH (11).

DPP-IV and ACE are vital membrane peptidases expressed physiologically by many tissues, such as the intestine (43, 44). As a matter of fact, human intestinal Caco-2 cells offer a dependable model that has already been established and confirmed for the investigation of peptides possessing DPP-IV or ACE inhibitory characteristics (45–47). Our findings indicated that CH maintains its ability to modulate the activity of both DPP-IV and ACE on Caco-2 cells, even though both hydrolysates are active in the range of concentration between 0.1 and 5 mg/mL as a function of the time (Figure 4A). In particular, CH inhibits both enzymes activity by 61.10 ± 1.70% and 76.90 ± 4.47% at the concentration of 5 mg/mL after 6 h of treatment (Figures 4,5), versus untreated cells, respectively, indicating that CH is less active in cell-based than in cell-free conditions. Similar results have been previously obtained on SH, PH, and peptic and tryptic hydrolysates of Arthrospira platensis (Spirulina) and Chlorella pyrenoidosa proteins, respectively (11, 45, 46, 48, 49). Also in these cases, the tested hydrolysates showed greater activity in cell-free assays than in cell-based assays. More in details, PH and SH inhibited the cellular DPP-IV activity by 53.6 ± 2.3% and 48.69 ± 7.29%, respectively, and the cellular ACE activity by 82.3 ± 4.2% and 74.6 ± 4.9%, respectively, at 5 mg/mL after 3 h (11). In addition, C. pyrenoidosa hydrolysates obtained using pepsin (CP) and trypsin (CT) inhibited the cellular ACE activity by 61.5 ± 7.7% and 69.9 ± 0.8%, respectively, at 5 mg/mL. When tested at the same concentration, they reduced the cellular DPP-IV activity by 38.4 and 42.5%, respectively (45).

This discrepancy in efficacy may be explained by the metabolic capabilities of Caco-2 cells, which express active proteases and peptidases that can actively metabolize peptides and impact their bioactivity (45). Thus, the intestine performs a dynamic function, not only in the process of vital nutrient absorption but also in actively modulating the physicochemical and biological profiles of food protein hydrolysates. Moreover, the peptides composition of CH here investigated results in line with a recent research dealing with the identification of novel casein-derived bioactive peptides and their potential (ACE)-inhibitory, antioxidant, and DPP-IV inhibitory activity when casein were fermented with Lactobacillus helveticus (50). Some of the identified peptides, i.e., DIPNPIGSE, EDVPSER, FPKYPVE are common to those generated by the fermentation.

For the first time, we have characterized the multifunctional behavior of a commercial casein hydrolysate, demonstrating its dual inhibitory properties on DPP-IV and ACE, using both cell-free and cell-based assays. Indeed, the active intestinal peptidases, which are expressed on cellular membrane, modulate peptide profile without impair their pleotropic activity. Hence, our findings constitute a significant foundation for future investigation of CH by in vivo and clinical studies for confirming its multifunctional and pleotropic behavior. With its ability to confer both hypotensive and antidiabetic benefits, CH may be a highly promising ingredient for use in the development of functional foods and dietary supplements aimed at preventing cardiovascular disease and metabolic syndrome.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

CL, JL, CB, GA, MB, GB, and FR: experiments. CL: discussion of the results. CL and JL: manuscript writing (original draft). CL and AA: manuscript editing. All authors have read and agreed to the published version of the manuscript.

This research and the APC were funded by A. Costantino & C. Spa.

FR was employed by A. Costantino & C. Spa which had the following involvement in the study: provided the calculated molecular weight distribution of peptide samples.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.