Inhibition of the in vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates

The aim of this work was to produce yellow field pea protein-derived peptides as inhibitors of α-amylase, α-glucosidase and pancreatic lipase activities. A pea protein concentrate was hydrolysed with alcalase, chymotrypsin, pepsin or trypsin and the hydrolysates separated into different fractions (<1, 1–3, 3–5, 5–10 kDa) by membrane ultrafiltration. Peptide sequence analysis showed that the alcalase hydrolysate had higher levels of di- and tripeptides when compared with the chymotrypsin, pepsin and trypsin hydrolysates. The peptide fractions inhibited α-amylase and α-glucosidase activities at levels that were similar to the unfractionated hydrolysates. The peptides were more active against α-amylase (inhibition at μg level) than α-glucosidase (mg level). In contrast, the fractionated peptides had reduced ability (IC₅₀ >4.2 mg mL⁻¹) when compared with the unfractionated hydrolysate (IC₅₀ <4.2 mg mL⁻¹) to inhibit lipase activity. Enzyme kinetic studies revealed that the peptides reduced α-amylase activity through competitive inhibition. However, inhibition of α-glucosidase activity was non-competitive.     

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identi?ed from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography–electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to β-lactoglobulin 1 f(71–77) and β-lactoglobulin 1 f(143–155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 μM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes.     

Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates

Microwave pretreatment and hydrolysis were applied to rainbow trout (Oncorhynchus mykiss) by-products to produce bioactive peptides with dual in vitro angiotensin-I converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV) inhibitory activities. Peptides were fractionated using the single step electrodialysis with ultrafiltration membrane (EDUF). Concentration of cationic peptides (CP) increased in the recovery solution, reaching 125 μg mL⁻¹ after a 4-h treatment with migration rate of 15.68 ± 2.98 g m⁻² h. CP fractions displayed ACE and DPP-IV I inhibitory properties, with IC₅₀ values of 0.0036 mg mL⁻¹ and 1.23 mg mL⁻¹ respectively. The bioactivity was attributed to the low molecular weight peptides (300–500 Da) recovered. CP exhibited non-competitive inhibition patterns for ACE and DPP-IV, which were dose dependent. These results showed that bioactive peptides can successfully be separated from complex hydrolysate mixtures by EDUF. The fractionated peptides can serve as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Bioactive protein hydrolysates in the functional food ingredient industry: Overcoming current challenges

Meat proteins and associated by-products can be used as a source of bioactive hydrolysates and peptides with potential for use as functional food ingredients. Functional foods are foods that have a potentially positive effect on health, beyond basic nutrition. Numerous bioactive peptides, including angiotensin-I-converting enzyme (ACE-I, EC 3.4.15.1) and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) inhibitors, have been generated from meat by-product proteins to date. However, in order to use and commercialize bioactive hydrolysates and peptides as food ingredients, a number of significant challenges must first be overcome. This article gives an overview of the current state-of-the-art of meat-derived bioactive hydrolysate and peptide uses in the food industry. It also reviews frequent challenges faced when developing biologically active hydrolysates and peptides as food ingredients. These challenges include, but are not limited to, high production costs, negative sensory attributes in end products, taste modifications of carrier food products and compliance with, for example, the European Food Safety Authority (EFSA), the Food and Drug Administration (FDA), and other regulatory bodies in China, or Japan, as well as potential toxicity or allergenicity. We suggest strategies that may assist in overcoming these challenges, focusing on those that may be used to improve the taste attributes of the end products.     

Antioxidant activities and functional properties of soy protein isolate hydrolysates obtained using microbial proteases

Soy protein isolate (SPI) hydrolysates were prepared using microbial proteases to produce peptides with antioxidant activity. The process parameters (substrate and enzyme concentrations), hydrolysis time, functional properties and the effects of ultrafiltration were further investigated. The results showed that the soy protein isolate exhibited a 7.0‐fold increase in antioxidant activity after hydrolysis. The hydrolysis parameters, defined by the experimental design, were a substrate concentration of 90 mg mL^?1 and the addition of 70.0 U of protease per mL of reaction. The maximum antioxidant activities were observed between 120 and 180 min of hydrolysis, where the degree of hydrolysis was approximately 20.0%. The hydrolysis increased solubility of the soy protein isolate; however, the hydrolysates exhibited a tendency to decrease in the interfacial activities and the heat stability. The SPI hydrolysates fractions obtained by ultrafiltration showed that the enzymatic hydrolysis resulted in samples with homogenous size and strong antioxidant activity.     

Comparison of volumetric and surface decontamination techniques for innovative processing of mealworm larvae (Tenebrio molitor)

For food and feed safety of edible insects, effective decontamination methods need to be evaluated and developed. Traditional decontamination and preparation methods were reviewed and thermal and innovative inactivation methods for the decontamination of mealworm larvae were evaluated and compared. The impact of the surface decontamination techniques direct and indirect plasma treatment, and of volumetric methods such as high hydrostatic pressure treatment (400, 500, and 600 MPa) and thermal treatments (45 °C and 90 °C) for up to 15 min on the surface microbial load and on the overall microbial count of mealworm larvae (Tenebrio molitor) have been investigated. It was found that the indirect plasma treatment was an effective means for the surface decontamination of mealworm larvae, whereas high hydrostatic pressure at 600 MPa and thermal treatments in a water bath at 90 °C in comparison resulted in the highest reduction of the overall count. It is thus concluded that volumetric methods are favorable for the inactivation of the gut microbiota of insects.Industrial relevanceEdible insects represent a valuable alternative protein source that could contribute to food and feed security and are industrially mainly unexploited. For a successful marketing of edible insects food and feed safety has to be ensured and effective decontamination methods need to be developed.     

Antioxidant activity of protein hydrolysates and purified peptides from Zizyphus jujuba fruits

In recent years, there has been a considerable interest in bioactive peptides derived from food proteins which might have beneficial effects on human health. Zizyphus jujuba is a medicinal plant with well-demonstrated biological functions for which various bioactive compounds except antioxidant peptides have been reported. Therefore, the aim of this study was to determine the antioxidant activity of Z. jujuba-derived protein hydrolysates and the purified peptides. Based upon this study, it was revealed that the hydrolysates prepared from Z. jujuba fruit possessed antioxidant effects. Among the prepared hydrolysates, trypsin hydrolysate with the highest antioxidant activity was fractionated using reverse-phase high-performance liquid chromatography (RP-HPLC). The most potent antioxidant peptides, named fractions F3 and F6, identified as VGQHTR (MW: 678.36 ± 0.3 Da) and GWLK (MW: 482.27 ± 0.3 Da), respectively, using tandem mass spectrometry. This study demonstrated that the derived hydrolysates and the purified peptides from Z. jujuba proteins can prevent oxidative reactions and might be underutilised for food preservation and medicinal purposes. However, more detailed studies are required to explore their antioxidant abilities in vivo.     

Dual function peptides from pepsin hydrolysates of whey protein isolate

The aim of this study was to investigate the effect of pepsin hydrolysates of whey protein isolate (WPI) on vascular relaxation and emulsifying capacity. WPI was subjected to pepsin hydrolysis for 5 h. The chromatographic profiles of the samples showed the formation of a wide variety of peptides. Addition of WPI hydrolysates in phenylephrine-contracted rat aortic rings induced a similar concentration-dependent relaxation in both endothelium-intact and endothelium-denuded preparations. In endothelium-denuded vessels the maximum relaxation induced by WPI fractions increased along the time, reaching over 70% after 3 h-hydrolysis on. In addition, the vascular relaxation was not associated with an inhibition of the angiotensin-converting enzyme or activation of K⁺ channels. Hydrolysed fractions were further evaluated for the emulsifying capacity (EC) and all tested fractions were able to keep an EC over 60%. These results reinforce the potential of WPI pepsin-hydrolysates as an option in the search for dual function peptides from whey proteins.     

The effect of time and origin of harvest on the in vitro biological activity of Palmaria palmata protein hydrolysates

The effect of starting protein composition, and the origin and time of harvesting on the in vitro tyrosinase, dipeptidyl peptidase (DPP) IV and angiotensin converting enzyme (ACE) inhibitory and antioxidant activity of Palmaria palmata protein hydrolysates were investigated. Electrophoretic profiles showed significant differences in aqueous protein extracts obtained from the red macroalga Palmaria palmata harvested at different times of the year. No significant difference was observed in aqueous protein profiles extracted from wild and aquacultured samples of Palmaria palmata. Protein extracts from Palmaria palmata samples harvested from wild plants in April, July and October, and samples cultivated on longlines and harvested in April were hydrolysed with Alcalase 2.4 L and Corolase PP. The hydrolysates, when tested at 10 mg/ml, were shown to inhibit tyrosinase by 37–56%. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) values of the hydrolysates ranged from 323–494 and 8.9–19.9 μmol trolox equivalents per gram, respectively. The Palmaria palmata hydrolysates inhibited DPP-IV (IC₅₀: 1.60–4.24 mg/mL) and ACE (IC₅₀: 0.14–0.35 mg/mL) activities. The starting protein composition had a significant effect on the tyrosinase inhibitory activity, while protein composition and hydrolytic enzyme preparation had a significant effect on DPP-IV inhibitory and antioxidant activities. In general, the origin of the samples, wild or cultivated, had no effect on the in vitro biological activity of the protein hydrolysates. The results show that Palmaria palmata hydrolysates may have potential applications as health enhancing ingredients and as food preservatives due to their antioxidant and tyrosinase inhibitory effects.     

The potential application of the protein hydrolysates of three medicinal plants: cytotoxicity and functional properties

Functional evaluation of encrypted bioactive peptides in protein structure helps to better understand those for using in pharmacy and food sciences. For this purpose, the total protein was extracted from Matricaria chamomilla, Ziziphora clinopodioides, and Cressa cretica, and partially purified with ammonium sulfate. Protein hydrolysates were obtained from pancreatin hydrolysis for 240 min and the enzyme hydrolysis was confirmed using the determination of hydrolysis degree and Fourier transform infrared (FT-IR) followed by the physicochemical and sensory properties were investigated. The results showed that all hydrolysates had both cytotoxic and antioxidant activities. Specifically, C. cretica hydrolysates represented cytotoxic activity against the MCF-7 cell line with the IC₅₀ of 135.21 µg/mL, while showed no significant growth inhibition effect on the HEK293 cell line. Besides, M. chamomilla hydrolysates showed the lowest bitterness value (1.125 ± 0.52). From the perspective of color investigation, M. chamomilla hydrolysates indicated the highest L^* and the lowest a^* factors. The highest turbidity and surface tension, and 10-fold more cancer cell killing effect under gastrointestinal digestion conditions were observed for M. chamomilla hydrolysates. Therefore, bioactive peptides might be formulated in designing of novel anticancer drugs or could be used in promising protocols for the production of food products with beneficial health effects.     

Peptic hydrolysis of bovine beta‐lactoglobulin under microwave treatment reduces its allergenicity in an ex vivo murine allergy model

In this study, the in vivo allergenicity of bovine beta‐lactoglobulin (BLG) in peptic whey protein hydrolysates generated during microwave and conventional heating treatments was assessed. The allergenicity of the hydrolysates was explored by studying the reaction of the murine jejunum from previously immunised Balb/c mice to treated BLG in an Ussing chamber. Intestinal anaphylactic reactions after stimulation of the gut‐associated immune system are a good indicator of potential in vivo allergenicity of whey hydrolysates. Fifty‐two per cent of BLG was hydrolysed by pepsin after only 3 min of microwave irradiation at 200 watts (W), yet it remained intact under conventional heating. Far‐ and near‐UV circular dichroism spectra indicated significant changes in BLG secondary and tertiary structures with microwave irradiation at 200 W. Pepsin whey protein hydrolysates obtained with microwave irradiation at 200 W for 3 min did not stimulate secretion of chloride in the Ussing chamber, as shown by the intensity of the short current values recorded (27.86 μA cm^?2), compared to the conventional pepsin hydrolysates (68.21 μA cm^?2). This demonstrates the low allergenicity of whey protein hydrolysates generated in this manner. These results confirm that microwave treatment combined with peptic hydrolysis could be applied to produce low allergenicity milk peptides.     

Effect of enzymatic hydrolysis on molecular weight distribution,techno-functional properties and sensory perception of pea protein isolates

Pea protein isolate (Pisum sativum “Navarro”) was hydrolyzed with 11 proteolytic enzymes at different hydrolysis times (15, 30, 60, and 120 min) to improve techno-functional and sensory properties. The degree of hydrolysis and changes within the molecular weight distribution were used as indicators for a reduced allergenic potential. The highest degree of hydrolysis was reached by Esperase hydrolysates (9.77%) after 120 min of hydrolysis, whereas Chymotrypsin hydrolysates showed the lowest (1.81%). Hydrolysis with Papain, Trypsin, Bromelain, Esperase, Savinase, and Alcalase suggested an effective degradation of the 72 kDa-convicilin fraction. Papain and Trypsin hydrolysates showed a degradation of the 50 kDa-mature vicilin after 15 min of hydrolysis. Most hydrolysates showed a significant increase in protein solubility at pH 4.5 at all times of hydrolysis. Trypsin hydrolysates showed the highest foaming (2271%) and emulsifying (719 mL/g) capacities. The bitterness of the hydrolysates was strongly correlated (P < 0.05) with the degree of hydrolysis. In general, enzymatic hydrolysis improved techno-functional properties indicating their potential usage as food ingredients.Industrial relevanceDue to their high protein content, peas are becoming an attractive ingredient for the food industry. However, pea protein isolates are often characterized by poor techno-functional and sensory properties. Enzymatic hydrolysis is known to change the molecular weight distribution of proteins. Consequently, the techno-functional and immunogenic properties might be altered selectively. In this study, enzymatic hydrolysis was applied, resulting in highly functional pea protein hydrolysates with a hypothesized reduction of main allergens. The lower bitter perception highlights their high potential as valuable functional food ingredients.     

Inhibition of dipeptidyl peptidase IV (DPP-IV) by proline containing casein-derived peptides

Dipeptides with a C terminal Pro inhibit dipeptidyl peptidase IV (DPP-IV), a key enzyme in incretin hormone processing. It was hypothesised that tri- and tetrapeptides with a proline at the C-terminus may also be DPP-IV inhibitors. Therefore, an in silico hydrolysis approach was used to release short (4 ? amino acids) C terminal Pro peptides from the individual caseins which constitute Pro rich substrates. This was achieved using theoretical digestion of caseins with a prolyl oligopeptidase activity. Fifteen peptides were subsequently selected for in vitro DPP-IV inhibitory analysis. Stability of these peptides to gastrointestinal enzymes was also evaluated in silico and the predicted breakdown peptides were assessed for their DPP-IV inhibitory and antioxidant potential. New DPP-IV inhibitors were identified, the most potent being Phe-Leu-Gln-Pro (IC₅₀ 65.3 ± 3.5 μM). A low in vitro antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging) activity was also associated with the peptides studied. The strategy presented highlights the utility of employing an in silico approach for the prediction of food-derived peptides with a potential role in glycaemic management for subsequent development of functional foods.     

Evaluation of the potential of dietary proteins as precursors of dipeptidyl peptidase (DPP)-IV inhibitors by an in silico approach

Synthetic inhibitors against the enzyme dipeptidyl peptidase (DPP)-IV, a serine protease implicated in the inactivation of incretin hormones, are currently used for the management of type 2 diabetes. The aim of the present study was to evaluate the potential of dietary proteins from various food commodities to serve as precursors of DPP-IV inhibitors by using an in silico approach. A total of 2256 fragments with peptide sequences matching those reported in the literature to present DPP-IV inhibitory activity were found in the 34 proteins that were investigated. Among those, Gly-Ala, Gly-Pro and Pro-Gly were the most frequently occurring sequences. Caseins from cow’s milk and collagens from bovine meat and salmon appeared to be the richest potential sources of DPP-IV inhibitors, whereas proteins from oat showed lower occurrence frequency values. This study suggests that dietary proteins could be used for generating DPP-IV inhibitory peptides.     

Reducing and radical-scavenging activities of whey protein hydrolysates prepared with Alcalase

Antioxidant activities of whey protein isolate (WPI) hydrolysates prepared by Alcalase treatment at different concentrations and times were investigated. The antioxidant activity of WPI hydrolysates, indicated by peroxide value and thiobarbituric acid-reactive substance values in a liposome-oxidizing system, increased with increasing hydrolysis time up to 5 h (P < 0.05). The WPI hydrolysates also showed greater radical-scavenging ability, greater Cu²⁺-chelating ability and improved reducing power when compared with non-hydrolysed WPI (P < 0.05). An increase in protein concentration was shown to significantly enhance antioxidant activities (P < 0.05). Although non-hydrolysed WPI displayed an antioxidative effect, it was far less potent than the hydrolysed WPI. This study shows that enzyme-hydrolysed WPI can act as a hydrogen donor, a metal ion chelator, and a radical stabiliser to inhibit lipid oxidation. The WPI hydrolysates produced by Alcalase could be employed in the food industry as an antioxidant to replace synthetic antioxidants.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

Inhibition of frozen storage‐induced oxidation and structural changes in myofibril of common carp (Cyprinus carpio) surimi by cryoprotectant and hydrolysed whey protein addition

The objective of the present study was to evaluate the efficacy of combined cryoprotectants (sucrose + sorbitol) and whey protein isolate (WPI) hydrolysates to inhibit protein oxidation and quality loss in common carp (Cyprinus carpio) surimi during frozen storage at ?25 °C. With increasing storage time, the carbonyl content of myofibrillar proteins increased from 4.02 nmolmg^‐1 protein (0 day) to 7.25, 6.31, 5.26 and 4.83 nmol mg^?1 protein (180 days; P < 0.05) for the control and samples with cryoprotectants, with cryoprotectants + WPI hydrolysates and with cryoprotectants + propyl gallate, respectively; protein surface hydrophobicity and turbidity increased in a similar trend, while sulfhydryl content, Ca‐ATPase activity, protein solubility and protein thermal stability decreased (P < 0.05). These results suggest that treatments with combined cryoprotectants and antioxidative WPI hydrolysates offer an effective approach to reducing the extent of protein oxidation in common carp surimi, thereby limiting protein structural changes known to impair texture of surimi products.     

Improving antioxidant activity of black bean protein by hydrolysis with protease combinations

This study explored the use of a simplex centroid design to produce protein hydrolysates with antioxidant properties using Alcalase^® 2.4L, Flavourzyme^® 500L and Neutrase^® 0.8L. Proteases kinetic parameters and the ultrafiltration of protein hydrolysates were also investigated. The highest antioxidant activity, in the studied conditions, was reached when the mixture of Alcalase^® 2.4L and Flavourzyme^® 500L was used in the hydrolysates production. The antioxidant power of the black bean proteins, measured by the total antioxidant capacity and reducing power assay, increased after hydrolysis by 31% and 70%, respectively. The black bean proteins hydrolysates fractions (3–30 kDa) showed an antioxidant activity decrease along with a reduction in molecular weight, demonstrating that a set of varied molecular weight peptides was responsible for the antioxidant characteristics of black bean protein hydrolysates.     

Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease involved in the degradation and inactivation of incretin hormones that act by stimulating glucose-dependent insulin secretion after meal ingestion. DPP-IV inhibitors have emerged as new and promising oral agents for the treatment of type 2 diabetes. The purpose of this study was to investigate the potential of β-lactoglobulin as natural source of DPP-IV inhibitory peptides. A whey protein concentrate rich in β-lactoglobulin was hydrolysed with trypsin and fractionated using a chromatographic separation at semipreparative scale. Two of the six collected fractions showed notable DPP-IV inhibitory activity. These fractions were analysed by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) to identify peptides responsible for the observed activity. The most potent fragment (IPAVF) corresponded to β-lactoglobulin f(78–82) which IC₅₀ value was 44.7 μM. The results suggest that peptides derived from β-lactoglobulin would be beneficial ingredients of foods against type 2 diabetes.