Evaluation of the multifunctional dipeptidyl-peptidase IV and angiotensin converting enzyme inhibitory properties of a casein hydrolysate using cell-free and cell-based assays.

The objective of the study was the evaluation of the potential pleiotropic effect of a commercial casein hydrolysate (CH). After an analysis of the composition, the BIOPEP-UWM database suggested that these peptides contained numerous sequences with potential inhibitory activities on angiotensin converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV). The anti-diabetic and anti-hypertensive effects of these peptides were thus assessed using either cell-free or cell-based assays. In the cell-free system, CH displayed inhibitory properties against DPP-IV (IC50 value equal to 0.38 ± 0.01 mg/mL) and ACE (IC50 value equal to 0.39 ± 0.01 mg/mL). Further, CH reduced the DPP-IV and ACE activities expressed by human intestinal Caco-2 cells by 61.10 ± 1.70% and 76.90 ± 4.47%, respectively, versus untreated cells, after 6 h of treatment at the concentration of 5 mg/mL. This first demonstration of the multifunctional behavior of this material suggests that it may become an anti-diabetic and/or anti-hypertensive ingredient to be included in the formulation of different functional food or nutraceutics.

Rainbow Trout (Oncorhynchus mykiss) as Source of Multifunctional Peptides with Antioxidant, ACE and DPP-IV Inhibitory Activities.

The present study aimed at characterizing the possible biological activities of the multifunctional low molecular weight fractions (<3 kDa) peptides isolated from rainbow trout (Oncorhynchus mykiss) obtained by enzymatic hydrolysis. The fish protein hydrolysate (FPH) was tested for its antioxidant property along with its angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. In particular, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), the ferric reducing antioxidant power (FRAP), the oxygen radical absorbance capacity (ORAC) assay and the 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays were carried out for the evaluation of the in vitro antioxidant activity. The cell-free ACE and DPP-IV inhibitory activity assays were also estimated, showing a dose-dependent inhibition. These biological properties were additionally quantified at the cellular level using human intestinal Caco-2 cells. Namely, the antioxidant activity was determined by evaluating the capability of the hydrolysate to reduce the H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels, and the DPP-IV activity assays show a reduction of enzyme activity of up to 27.57 ± 3.7% at 5 mg/mL. The results indicate that Oncorhynchus mykiss-derived peptides may have potential employment as health-promoting ingredients.

Isolation and functional characterization of hemp seed protein-derived short- and medium-chain peptide mixtures with multifunctional properties for metabolic syndrome prevention.

This study aims to obtain a valuable mixture of short-chain peptides from hempseed as a new ingredient for developing nutraceutical and functional foods useful for preventing metabolic syndrome that represents the major cause of death globally. A dedicated analytical platform based on a purification step by size exclusion chromatography or ultrafiltration membrane and high-resolution mass spectrometry was developed to isolate and comprehensively characterize short-chain peptides leading to the identification of more than 500 short-chain peptides. Our results indicated that the short-chain peptide mixture was about three times more active than the medium-chain peptide mixture and total hydrolysate with respect to measured inhibition of the angiotensin-converting enzyme. The short-chain peptide mixture was also two times more active as a dipeptidyl peptidase IV, and twofold more active on the cholesterol metabolism pathway through the modulation of low-density lipoprotein receptor.

Gel-Forming of Self-Assembling Peptides Functionalized with Food Bioactive Motifs Modulate DPP-IV and ACE Inhibitory Activity in Human Intestinal Caco-2 Cells.

Food bioactive peptides are increasingly used for formulating food products, nutraceuticals, and functional food, since they are generally considered safe for human consumption and metabolic syndrome prevention. They are also becoming popular as sustainable sources of novel functional biomaterials such as hydrogels, edible nanonutraceuticals, delivery systems, and packing materials. However, such food peptides are mostly unstable, and degrade during food processing, or in a gastrointestinal environment, thus resulting in low bioavailability precluding their practical applications. Here, we decided to functionalize the well-known and characterized self-assembling peptide RADA16 with two synthetic analogues of food bioactive peptides deriving from the hydrolysis of soybean glycinin and lupin ß-conglutin (namely IAVPTGVA and LTFPGSAED) for control of and improvement in their gel-forming nanostructures, biomechanics, and biological features. Extensive characterization was performed via Circular Dichroism (CD) spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), Thioflavin T (ThT) binding assay, rheological measurements, and Atomic Force Microscopy (AFM) analysis. Lastly, since self-assembling peptides (SAPs) can be co-assembled with diluent SAPs (without a bioactive epitope) as an approach to control the density of biological signals and therefore attain enhanced bioactivity, we investigated the effect of the co-assembly of RADA16 and functionalized food bioactive SAPs (dubbed cAP-Soy1 and cAP-Lup1) for the growth of Caco-2 human intestinal cells and contextually we characterized their biological activities as DPP-IV and ACE inhibitors, in order to demonstrate their potential use for the prevention of metabolic syndrome.

Mechanistic Insights into Angiotensin I-Converting Enzyme Inhibitory Tripeptides to Decipher the Chemical Basis of Their Activity.

Food proteins are an important source of bioactive peptides, and the angiotensin I-converting enzyme (ACE) inhibitors are worthy of attention for their possible beneficial effects in subjects with mild hypertension. However, the chemical basis underpinning their activity is not well-understood, hampering the discovery of novel inhibitory sequences from the plethora of peptides encrypted in food proteins. This work combined computational and in vitro investigations to describe precisely the chemical basis of potent inhibitory tripeptides. A substantial set of previously uncharacterized tripeptides have been investigated in silico and in vitro, and LCP was described for the first time as a potent ACE inhibitory peptide with IC50 values of 8.25 and 6.95 µM in cell-free and cell-based assays, respectively. The outcomes presented could serve to better understand the chemical basis of already characterized potent inhibitory tripeptides or as a blueprint to design novel and potent inhibitory peptides and peptide-like molecules.

Integrated Evaluation of the Multifunctional DPP-IV and ACE Inhibitory Effect of Soybean and Pea Protein Hydrolysates.

Nowadays, notwithstanding their nutritional and technological properties, food bioactive peptides from plant sources garner increasing attention for their ability to impart more than one beneficial effect on human health. Legumes, which stand out thanks to their high protein content, represent valuable sources of bioactive peptides. In this context, this study focused on the characterization of the potential pleotropic activity of two commercially available soybean (SH) and pea (PH) protein hydrolysates, respectively. Since the biological activity of a specific protein hydrolysate is strictly correlated with its chemical composition, the first aim of the study was to identify the compositions of the SH and PH peptides. Peptidomic analysis revealed that most of the identified peptides within both mixtures belong to storage proteins. Interestingly, according to the BIOPEP-UWM database, all the peptides contain more than one active motive with known inhibitory angiotensin converting enzyme (ACE) and dipeptidyl-dipeptidases (DPP)-IV sequences. Indeed, the results indicated that both SH and PH 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. In addition, both hydrolysates reduced the activity of DPP-IV and ACE enzymes which are expressed on the surface of human intestinal Caco-2 cells. These findings clearly support that notion that SH and PH may represent new ingredients with anti-diabetic and hypotensive effects for the development of innovative multifunctional foods and/or nutraceuticals for the prevention of metabolic syndrome.

Preparation, Characterization and In Vitro Stability of a Novel ACE-Inhibitory Peptide from Soybean Protein.

A soy protein isolate was hydrolyzed with Alcalase®, Flavourzyme® and their combination, and the resulting hydrolysates (A, F and A + F) were ultrafiltered and analyzed through SDS-PAGE. Fractions with MW < 1 kDa were investigated for their ACE-inhibitory activity, and the most active one (A < 1 kDa) was purified by semi-preparative RP-HPLC, affording three further subfractions. NMR analysis and Edman degradation of the most active subfraction (A1) enabled the identification of four putative sequences (ALKPDNR, VVPD, NDRP and NDTP), which were prepared by solid-phase synthesis. The comparison of their ACE-inhibitory activities suggested that the novel peptide NDRP might be the main agent responsible for A1 fraction ACE inhibition (ACE inhibition = 87.75 ± 0.61%; IC50 = 148.28 ± 9.83 µg mL−1). NDRP acts as a non-competitive inhibitor and is stable towards gastrointestinal simulated digestion. The Multiple Reaction Monitoring (MRM) analysis confirmed the presence of NDRP in A < 1 kDa.

Legumes are valuable sources of tocopherols.

Grain legumes contain numerous phytochemicals useful for their nutritional or nutraceutical properties, such as tocopherols, involved in the prevention of cardiovascular disease and eye pathologies. In this work, tocopherols were quantified in soybean, chickpea, lentil, pea, common bean, broad bean, and three lupin species. In all samples, the gamma congener was the most abundant tocopherol, followed by minor quantities of alpha-tocopherol (with the exception of common bean lacking in this congener) and delta-tocopherol (with the exception of Lupinus angustifolius and Lupinus mutabilis). Beta-tocopherol and tocotrienols were never detected. Some samples of soybean, pea, white lupin and chickpea contained over 10mg/100g seeds of total tocopherols. In order to estimate the nutritional value, the vitamin E activity was calculated. Chickpea, soybean and, to a lesser extent, lupin, broad bean and pea may contribute in a relevant way to the daily intake of this vitamin.

Composition of the Protein Ingredients from Insoluble Oat Byproducts Treated with Food-Grade Enzymes, Such as Amylase, Cellulose/Xylanase, and Protease.

The manufacture of plant-based drinks has the drawback of a huge production of underexploited press cakes. In particular, the oat press cake is mainly used in feed formulation, whereas added-value applications in human nutrition are scarce. Considering that enzymatic treatments may be useful to improve the nutritional quality of these insoluble byproducts, this study aimed to evaluate whether the treatment with some food-grade enzymes, such as amylase, cellulase/xylanase, protease, and their combination, may be useful to achieve this goal. Proteomic and peptidomic studies showed that the enzymatic treatments improved the protein extraction yields and induced a release of low molecular weight (LMW) peptides that were demonstrated to provide a useful antioxidant activity. In the treated oat press cake proteins, the concentration of the bound phenolic compounds was decreased, with the exception of caffeic acid, which was increased, and avenanthramides, which remained unchanged. Finally, the enzymatic treatment decreased the concentration of phytic acid. All these results indicate that the enzymatic treatments may be useful to ameliorate the nutritional profile of these protein ingredients, before their inclusion in different food products.

A heuristic, computer-driven and top-down approach to identify novel bioactive peptides: A proof-of-principle on angiotensin I converting enzyme inhibitory peptides.

Bioactive peptides are short peptides (3-20 amino acid residues in length) endowed of specific biological activities. The identification and characterization of bioactive peptides of food origin are crucial to better understand the physiological consequences of food, as well as to design novel foods, ingredients, supplements, and diets to counteract mild metabolic disorders. For this reason, the identification of bioactive peptides is also relevant from a pharmaceutical standpoint. Nevertheless, the systematic identification of bioactive sequences of food origin is still challenging and relies mainly on the so defined "bottom-up" approaches, which rarely results in the total identification of most active sequences. Conversely, "top-down" approaches aim at identifying bioactive sequences with certain features and may be more suitable for the precise identification of very potent bioactive peptides. In this context, this work presents a top-down, computer-assisted and hypothesis-driven identification of potent angiotensin I converting enzyme inhibitory tripeptides, as a proof of principle. A virtual library of 6840 tripeptides was screened in silico to identify potential highly potent inhibitory peptides. Then, computational results were confirmed experimentally and a very potent novel sequence, LMP was identified. LMP showed an IC50 of 15.8 and 6.8 µM in cell-free and cell-based assays, respectively. In addition, a bioinformatics approach was used to search potential food sources of LMP. Yolk proteins were identified as a possible relevant source to analyze in further experiments. Overall, the method presented may represent a powerful and versatile framework for a systematic, high-throughput and top-down identification of bioactive peptides.

Investigation of Chlorella pyrenoidosa Protein as a Source of Novel Angiotensin I-Converting Enzyme (ACE) and Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides.

Chlorella pyrenoidosa (C. pyrenoidosa) is a microalgae species with a remarkably high protein content that may potentially become a source of hypotensive and hypoglycemic peptides. In this study, C. pyrenoidosa proteins were extracted and hydrolyzed overnight with pepsin and trypsin with final degrees of hydrolysis of 18.7% and 35.5%, respectively. By LC-MS/MS, 47 valid peptides were identified in the peptic hydrolysate (CP) and 66 in the tryptic one (CT). At the concentration of 1.0 mg/mL, CP and CT hydrolysates inhibit in vitro the angiotensin-converting enzyme (ACE) activity by 84.2 ± 0.37% and 78.6 ± 1.7%, respectively, whereas, tested at cellular level at the concentration of 5.0 mg/mL, they reduce the ACE activity by 61.5 ± 7.7% and 69.9 ± 0.8%, respectively. At the concentration of 5.0 mg/mL, they decrease in vitro the DPP-IV activity by 63.7% and 69.6% and in Caco-2 cells by 38.4% and 42.5%, respectively. Short peptides (≤10 amino acids) were selected for investigating the potential interaction with ACE and DPP-IV by using molecular modeling approaches and four peptides were predicted to block both enzymes. Finally, the stability of these peptides was investigated against gastrointestinal digestion.

Analysis of Narrow-Leaf Lupin Proteins in Lupin-Enriched Pasta by Untargeted and Targeted Mass Spectrometry.

The supplementation of different food items with grain legumes and, in particular, with lupin has been demonstrated to provide useful health benefits, especially in the area of cardiovascular disease prevention. In this work, label free quantitative untargeted and targeted approaches based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for investigating the protein profile of three pasta samples containing different percentages of narrow-leaf lupin flour were carried out. The untargeted method permitted the identification of the main acidic globulins (α-conglutin, ß-conglutin, and δ-conglutin) and the comparison of their profile with raw lupin flour. The targeted method, based on High-performance liquid chromatography electrospray ionization tandem mass spectrometry HPLC-Chip-Multiple Reaction Monitoring (MRM) mode, allowed the quantification of γ-conglutin, the main hypoglycemic component of lupin protein: its concentration was around 2.25 mg/g in sample A, 2.16 mg/g in sample D, and 0.57 mg/g in sample F.

Assessment of the Multifunctional Behavior of Lupin Peptide P7 and Its Metabolite Using an Integrated Strategy.

LTFPGSAED (P7) is a multifunctional hypocholesterolemic and hypoglycemic lupin peptide. While assessing its angiotensin-converting enzyme (ACE) inhibitory activity, it was more effective in intestinal Caco-2 cells (IC50 of 13.7 µM) than in renal HK-2 cells (IC50 of 79.6 µM). This discrepancy was explained by the metabolic transformation mediated by intestinal peptidases, which produced two main detected peptides, TFPGSAED and LTFPG. Indeed LTFPG, dynamically generated by intestinal dipeptidyl peptidase IV as well as its parent peptide P7 were linearly absorbed by mature Caco-2 cells. An in silico study demonstrated that the metabolite was a better ligand of the ACE enzyme than P7. These results are in agreement with an in vivo study, previously performed by Aluko et al., which has shown that LTFPG is an effective hypotensive peptide. Our work highlights the dynamic nature of bioactive food peptides that may be modulated by the metabolic activity of intestinal cells.

Recent Advances in Microalgae Peptides: Cardiovascular Health Benefits and Analysis.

There is now great interest in food protein hydrolysates and food-derived peptides, because they may provide numerous health benefits. Among other foodstuffs, microalgae appear to be sustainable sources of proteins and bioactive peptides that can be exploited in foods and functional formulations. This review considers protein hydrolysates and individual peptides that may be relevant in cardiovascular disease prevention because they mimic the functions of mediators involved in pathologic processes that represent relevant risk factors for cardiovascular disease development, such as hypercholesterolemia, hypertension, diabetes, inflammation, and oxidative status. Some of these peptides are also multifunctional (i.e., they offer more than one benefit). Moreover, the most efficient techniques for protein extraction and hydrolyzation are commented on, as well as the best methodologies for high-throughput detection and quantification. Finally, current challenges and critical issues are discussed.

Quinolizidine alkaloids in seeds of lupin genotypes of different origins.

The intake of lupin-based foods could imply the exposure of consumers to quinolizidine alkaloids. The objectives of this study were to assess the genetic variation among and within 11 geographic regions of Lupinus albus ecotypes, verify the quinolizidine alkaloids amount of alkaloid-poor L. albus and Lupinus angustifolius varieties, and assess the effect of two climatically contrasting Italian environments on the alkaloid content. The quantitation was performed by GC-MS, and in all samples lupanine was the most abundant quinolizidine alkaloid, followed by albine and 13alpha-hydroxylupanine for L. albus and by 13alpha-hydroxylupanine and angustifoline for L. angustifolius. Some regions tended to have a high (Azores) or low (Egypt, Near East, Maghreb) total alkaloids content, but the variation among ecotypes within regions was larger than that among regions following the estimation of variance components. Alkaloid-poor varieties tended to have higher total alkaloid contents when grown in the subcontinental climate site, exceeding in some cases the limit of 0.200 mg/g.

Effect of genotype and environment on fatty acid composition of Lupinus albus L. seed.

Six cultivars of Lupinus albus L. (white lupin) were grown in two subcontinental-climate environments and one Mediterranean-climate environment in Italy, to assess the influence of genotypic (G) and genotype×environment (GE) interaction effects on grain yield and grain content of oil, total saturated fatty acids (FAs), polyunsaturated FAs, monounsaturated FAs, and ω-3/ω-6 FA ratio. The variance of genotypic effects was much larger than the GE interaction variance for all variables, except for grain yield, indicating that oil content and FA composition of different varieties can be assessed reliably in just a few test environments. Gas-chromatographic analyses highlighted that linoleic acid and α-linolenic acid were in the range 1.76-4.76mg/g flour (7.79-15.81% of total FAs) and 1.17-3.14mg/g flour (5.40-10.36% of total FAs), respectively. As a consequence, the analysed lupin seeds exhibited a very favourable ω-3/ω-6 FA ratio, ranging from 0.49 to 0.79.

Parameters for the evaluation of the thermal damage and nutraceutical potential of lupin-based ingredients and food products.

Foods based on sweet lupin proteins are gaining attention from industry and consumers because of their possible role in the prevention of cardiovascular disease. When promoting lupin-based foods for inclusion in a daily diet, the thermal damage suffered during processing is of relevance to the bioactive and nutritional quality of the food product. N-(2-furoylmethyl)-L-lysine (furosine) quantification demonstrates that currently available sweet lupin protein isolates have a thermal damage comparable to or lower than other traditional food ingredients, and are a good source of lysine in non-dairy products. In lupin-based foods claiming to have cholesterol-lowering potential, shotgun proteomics offers itself as a fast and effective screening method for assessing the biological availability of active peptides. Such a method is readily applicable to other legume-enriched food products.

Hydrolytic degradation of azimsulfuron, a sulfonylurea herbicide.

The chemical degradation of the herbicide azimsulfuron was investigated in aqueous solutions at different pH values. The hydrolysis rate, determined by HPLC analyses, was pH dependent and was much faster in acidic than in neutral or weakly basic conditions. The metabolites formed at different pH values were compared with standards when possible or isolated and identified using ESI-LC-MS/MS, (1)H NMR and (13)C NMR. The two main products of hydrolysis in mild acidic solution were identified as 2-amino-4,6-dimethoxy-pyrimidine and 2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide, both produced as a result of the sulfonylurea bridge cleavage. Under basic conditions, a new product, a substituted 2-pyrimidinamine, deriving from the contraction of the sulfonylurea bridge, was isolated and completely characterized for the first time.

Exploration of Potentially Bioactive Peptides Generated from the Enzymatic Hydrolysis of Hempseed Proteins.

The seed of industrial hemp is an underexploited protein source. In view of a possible use in functional foods, a hempseed protein concentrate was hydrolyzed with pepsin, trypsin, pancreatin, or a mixture of these enzymes. A detailed peptidomic analysis using data-dependent acquisition showed that the numbers of peptides identified ranged from 90 belonging to 33 parent proteins in the peptic hydrolysate to 9 belonging to 6 proteins in the pancreatin digest. The peptic and tryptic hydrolysates resulted to be the most efficient inhibitors of 3-hydroxymethyl-coenzyme A reductase activity when tested on the catalytic domain of the enzyme. Using the open access tools PeptideRanker and BIOPEP, a list of potentially bioactive peptides was generated: the alleged activities included the antioxidant property, the glucose uptake stimulating activity, the inhibition of dipeptidyl peptidase-IV and angiotensin-converting enzyme I.

New ACE-Inhibitory Peptides from Hemp Seed (Cannabis sativa L.) Proteins.

A hemp seed protein isolate, prepared from defatted hemp seed meals by alkaline solubilization/acid precipitation, was subjected to extensive chemical hydrolysis under acid conditions (6 M HCl). The resulting hydrolysate was fractionated by semipreparative RP-HPLC, and the purified fractions were tested as inhibitors of angiotensin converting enzyme (ACE). Mono- and bidimensional NMR experiments and LC-MS analyses led to the identification of four potentially bioactive peptides, i.e. GVLY, IEE, LGV, and RVR. They were prepared by solid-phase synthesis, and tested for ACE-inhibitory activity. The IC50 values were GVLY 16 ± 1.5 µM, LGV 145 ± 13 µM, and RVR 526 ± 33 µM, confirming that hemp seed may be a valuable source of hypotensive peptides.