Strategies for the release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides in an enzymatic hydrolyzate of α-lactalbumin

Abstract

Bovine α-lactalbumin (α-La) contains numerous dipeptidyl peptidase IV (DPP-IV) inhibitory peptides sequences within its primary structure. In silico analysis indicated that the targeted hydrolysis of α-La with elastase should release DPP-IV inhibitory peptide sequences. An α-La isolate was hydrolysed with elastase under different conditions using an experimental design approach incorporating 3 factors (temperature, pH and enzyme to substrate ratio (E:S) ratio) at 2 levels. The hydrolyzate generated at pH 8.5, 50C, E:S 2.0% (w/w) (H9) displayed the lowest half maximal DPP-IV inhibitory concentration (IC50 = 1.20 ± 0.12 mg mL -1). Five α-La-derived DPP-IV inhibitory peptides (GY, GL, GI, NY and WL) predicted to be released in silico were identified by liquid-chromatography tandem mass spectrometry (LC-MS/MS) within H9 and its simulated gastrointestinal digestion (SGID) sample. This proof of concept study demonstrated the benefit of using a targeted approach combined with an experimental design in the generation of dietary protein hydrolyzates with DPP-IV inhibitory properties. IntroductionDipeptidyl peptidase IV (DPP-IV) is an ubiquitous enzyme which is responsible for the cleavage and inactivation of the incretin hormones, glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) 1 . In the context of type 2 diabetes management, DPP-IV inhibition may be used as a means to improve the regulation of serum glucose in humans. Various DPP-IV inhibitory drugs (gliptins), have been developed to maintain the insulinotropic activity of the incretins in the post-prandial phase 2 .Dietary components, including food protein-derived peptides, have been shown to play a role in the inhibition of DPP-IV in vitro and in certain instances in vivo, for reviews, see: 3,4,5 . In silico studies have shown that selected DPP-IV inhibitory peptide sequences may be found within a wide range of dietary proteins 6,7 . To date, milk proteins appear to be the most frequently studied substrate for the generation of DPP-IV inhibitory peptides. Depending on the enzymatic strategy employed, different milk proteins may appear to be more suitable substrates for the production of DPP-IV inhibitory peptides. For instance, it was recently predicted by Tulipano et al.8 using in silico digestion with gastrointestinal proteinases that -lactoglobulin (-Lg) should yield a higher number of previously identified DPP-IV inhibitory peptides than -La. This was further confirmed following in vitro digestion of -Lg and -La with gastrointestinal enzymes, yielding hydrolyzates with IC 50 values of 0.74 and 1.70 mg mL -1, respectively 8 . Other in silico analysis of the major milk proteins indicated that -lactalbumin (-La) displayed the highest level of sequence coverage (43.9%) for previously identified DPP-IV inhibitory peptides as well as the highest DPP-IV inhibitory potency index (i.e., 17.9 10 -6 µM -1 g -1 ) 7 . It has been suggested that discrepancies between studies to determine the most adequate substrate for the genera...

Dipeptidyl Peptidase-IV Inhibitory Activity and Related Molecular Mechanism of Bovine α-Lactalbumin-Derived Peptides

“…In the present study, bovine α-lactalbumin hydrolysates were obtained by alcalase, and the DPP-IV inhibitory activity was determined. Our results showed that 4 h hydrolysates possessed the strongest DPP-IV inhibitory activity with inhibition rate of 82.30 ± 1.39% at a concentration of 1.0 mg/mL (Figure 1), which was higher than that of bovine α-lactalbumin hydrolysate generated by elastase (with DPP-IV inhibition rate of 75.8 ± 3.7% at a concentration of 3.1 mg/mL) [24].…”

“…Among the two identified peptides shown in Table 1 , peptide ILDKVGINY has been demonstrated with DPP-IV inhibitory activity previously (IC 50 values = 263 μM) [ 22 ]. As for peptide ELKDLKGY, although it has not been mentioned in the literature previously, it contains the same amino acid sequences with several reported DPP-IV inhibitory peptides, such as RELKDLKGY, RELKDLKGYG, RELKDLKGYGG, RELKDLKGYGGVS, RELKDLKGY, KDL, KGY, and GY [ 22 , 24 ]. Various studies showed that the peptides displaying potent DPP-IV inhibitory activity generally have a length of 2 to 7 amino acids [ 30 ].…”

“…Therefore, the utilization of α-lactalbumin makes great sense in the governance of environmental problems caused by whey by-products. Previous in silico studies have shown that bovine α-lactalbumin contain DPP-IV inhibitory peptide sequences, which was confirmed by the potent DPP-IV inhibitory activity of bovine α-lactalbumin hydrolysates and bovine α-lactalbumin derived peptides [20][21][22][23][24]. However, the underlying molecular mechanism of α-lactalbumin derived peptides on DPP-IV inhibition has not been understood clearly.…”

Features of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from dietary proteins

“…Other approaches are based on peptide cutters used in in silico digestion of selected food proteins to predict the release of target peptides (known DPP‐IV inhibitory peptides or peptides possessing features of DPP‐IV inhibitory peptides). Information generated by peptide cutters has been described during the hydrolysis of bovine α‐lactalbumin (α‐La) with porcine pancreatic elastase (Nongonierma, Le Maux, Hamayon, & FitzGerald, ). However, it was found that only 60% of the peptides predicted to be released in silico were actually identified by liquid chromatography tandem mass spectrometry (LC‐MS/MS) in the bovine α‐La hydrolysate.…”

Bovine α-Lactalbumin Hydrolysates (α-LAH) Ameliorate Adipose Insulin Resistance and Inflammation in High-Fat Diet-Fed C57BL/6J Mice

“…In Gottingen minipigs, bovine α-lactalbumin reduced serum haptoglobin and C-reactive protein level, resulting in the amelioration of Western diet-induced glucose intolerance [ 23 ]. Furthermore, peptides derived from bovine α-lactalbumin inhibited dipeptidyl peptidase 4 (DPP-4) activity, suppressed angiotensin converting enzyme (ACE) activity, scavenged 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonate] (ABTS + ) radical and stimulated glucagon-like peptide-2 secretion [ 24 , 25 , 26 , 27 , 28 ]. However, there are few studies about the efficacy and underlying mechanisms of bovine α-lactalbumin hydrolysates against insulin resistance.…”