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Dipeptidyl peptidase-IV (DPP-IV) inhibiting peptides have attracted increased attention because of their possible beneficial effects on glycemic homeostasis. However, the structural basis underpinning their activities has not been well understood. This study combined computational and in vitro investigations to explore the structural basis of DPP-IV inhibitory peptides. We first superimposed the Xaa-Pro-type peptide-like structures from several crystal structures of DPP-IV ligand-protein complexes to analyze the recognition interactions of DPP-IV to peptides. Thereafter, a small set of Xaa-Pro-type peptides was designed to explore the effect of key interactions on inhibitory activity. The intramolecular interaction of Xaa-Pro-type peptides at the first and third positions from the N-terminus was pivotal to their inhibitory activities. Residue interactions between DPP-IV and residues of the peptides at the fourth and fifth positions of the N-terminus contributed significantly to the inhibitory effect of Xaa-Pro-type tetrapeptides and pentapeptides. Based on the interaction descriptors, quantitative structure-activity relationship (QSAR) studies with the DPP-IV inhibitory peptides resulted in valid models with high R 2 values (0.33 for tripeptides; 0.68 for tetrapeptides and pentapeptides). Taken together, the structural information on DPP-IV and peptides in this study facilitated the development of novel DPP-IV inhibitory peptides.Q 2 values (0.33 for tripeptides; 0.68 for tetrapeptides and pentapeptides). Taken together, the structural information on DPP-IV and peptides in this study facilitated the development of novel DPP-IV inhibitory peptides.

The aim of this study was to investigate the dipeptidyl peptidase-IV (DPP-IV) inhibition and metabolic stability of a casein-derived peptide Val-Pro-Tyr-Pro-Gln (VPYPQ) and its fragments as well as their release from casein following hydrolysis. Results showed that VPYPQ was the most potent DPP-IV inhibitory peptide among them with an IC 50 value of 41.45 μM. This might be due to its two internal Pro residues at positions 2 and 4. Moreover, VPYPQ was resistant to hydrolysis by gastrointestinal enzymes and was relatively more stable to hydrolysis by DPP-IV and peptidases in plasma compared with its fragments. Additionally, oral administration of VPYPQ at a dose of 90 μmol/kg body weight could reduce the postprandial blood glucose levels in mice. More importantly, VPYPQ could be released efficiently from casein following hydrolysis by a combination of papain and in vitro digestion, reaching up to 3211.15 μg/g. Therefore, VPYPQ was a promising casein-derived DPP-IV inhibitor.

This study aimed to examine the feasibility of improving umami taste of peanut protein isolate hydrolysate (PPIH) and its fractions (obtained via ethanol precipitation and gel filtration chromatography) and peptide constituents, through their reactions with glucose at 98 °C for 70 min (Maillard reaction). To get insights into the characteristics of the peptide contributors with high umami intensity and/or umami-enhancing activity, Maillard reaction-guided and sensory-guided fractionation and characterization using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and eXpose algorithm calculation method were subsequently involved. Three umami and umami-enhancing peptides, three umami-enhancing peptides with other tastes, three bitter peptides and one astringent peptide were found. For the first time, peptide TP was reported to suppress umami intensity of monosodium glutamate (MSG) aqueous solution. Combined treatments of enzymatic hydrolysis and Maillard reaction on PPI facilitated umami enhancement. Maillard products of identified peptides had higher umami intensities and/or greater umami-enhancing abilities. Pyroglutamylproline (pyroEP) and glucosylated EP were discovered as the major compounds of EP Maillard product., (Copyright © 2018. Published by Elsevier Ltd.)

Size-exclusion chromatography (SEC) with wide application in peptide analysis presents challenges in determination of molecular weight distribution due to the relatively low resolution. In this study, a stop-flow reversed phase liquid chromatography (RPLC) × SEC system was constructed, aiming at improving the peptide separation in SEC. As the chromatographic dispersion during stop-flow operation might contribute to the band broadening in the first dimension (1st D) RPLC, the effects of different stop-flow operational parameters on the additional band broadening were quantitatively evaluated. Unlike analytes of large molecular size or long retention time with low effective diffusion coefficient (D eff ), additional band broadening was evidently observed for analytes of small molecular size and short retention time (high D eff ). Therefore, optimal flow rate, low column temperature and short stop-flow time were suggested for analyzing small molecules of short retention time. The established stop-flow two-dimensional liquid chromatography (2D-LC) was further tested on protein hydrolysates. The resolution was evidently improved for both heart-cutting and comprehensive 2D-LC analysis (despite additional band broadening in RPLC). Compared with heart-cutting analysis with higher 1st D resolution for selective fractions, comprehensive analysis could provide more complete information about the molecular weight distribution of the eluting solutes along RPLC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The need to improve the peak capacity of liquid chromatography motivates the development of two-dimensional analysis systems. This paper presented a fully automated stop-flow two-dimensional liquid chromatography system with size exclusion chromatography followed by reversed phase liquid chromatography (SEC×RPLC) to efficiently separate peptides. The effects of different stop-flow operational parameters (stop-flow time, peak parking position, number of stop-flow periods and column temperature) on band broadening in the first dimension (1 st D) SEC column were quantitatively evaluated by using commercial small proteins and peptides. Results showed that the effects of peak parking position and the number of stop-flow periods on band broadening were relatively small. Unlike stop-flow analysis of large molecules with a long running time, additional band broadening was evidently observed for small molecule analytes due to the relatively high effective diffusion coefficient (D eff ). Therefore, shorter analysis time and lower 1 st D column temperature were suggested for analyzing small molecules. The stop-flow two-dimensional liquid chromatography (2D-LC) system was further tested on peanut peptides and an evidently improved resolution was observed for both stop-flow heart-cutting and comprehensive 2D-LC analysis (in spite of additional band broadening in SEC). The stop-flow SEC×RPLC, especially heart-cutting analysis with shorter analysis time and higher 1 st D resolution for selected fractions, offers a promising approach for efficient analysis of complex samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The substrate specificity of aminopeptidases has often been determined against aminoacyl-p-nitroanilide; thus, its specificity toward synthetic peptides and complex substrates remained unclear. The hydrolysis specificity of an aminopeptidase from Bacillus licheniformis SWJS33 (BLAM) was evaluated using a series of synthetic peptides and soybean protein isolate. The aminopeptidase showed high specificity for dipeptides with Leu, Val, Ala, Gly, and Phe at the N-terminus, and the specificity was significantly affected by the nature of the penultimate residue. In the hydrolysis of soy protein isolate, BLAM preferred peptides with Leu, Glu, Gly, and Ala at the N-terminus by free amino acid analysis and preferred peptides with Leu, Ala, Ser, Trp, and Tyr at the N-terminus by UPLC-MS/MS. The introduction of complex substrates provides a deeper understanding of the aminopeptidase's specificity, which can instruct the application of the enzyme in protein hydrolysis.

Five tasty peptides were separated from soy sauce, by sensory-guided fractionation, using macroporous resin, medium-pressure liquid chromatography and reverse phase-high performance liquid chromatography, and identified by ultra-performance liquid chromatography tandem mass-spectrometry as ALPEEV, LPEEV, AQALQAQA, EQQQQ and EAGIQ (which originated from glycinin A1bB2-445, glycinin A1bB2-445, cobyric acid synthase, leucine-tRNA ligase and glycoprotein glucosyltransferase, respectively). LPEEV, AQALQAQA and EQQQQ tasted umami with threshold values of 0.43, 1.25 and 0.76mmol/l, respectively. ALPEEV and EAGIQ had minimal umami taste, but ALPEEV, EAGIQ and LPEEV showed umami-enhancement with a threshold estimated at 1.52, 1.94 and 3.41mmol/l, respectively. In addition, the synthetic peptides showed much better sensory taste than mixtures of their constitutive amino acids. It indicated that peptides might play an important role in the umami taste of soy sauce., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, the performance and separation characteristics of four macroporous resins for purifying umami peptides from soy sauce were examined. Results showed that the resins could separate the peptides of soy sauce, and the particle diffusion kinetics model was suitable for describing the whole exothermic (ΔH < 43 kJ/mol) adsorption process on the SP-825 and HP-20 resins, while the pseudo-second-order kinetics model accurately described the XAD-16 and HP-2 MGL resins. Furthermore, the adsorption processes of the peptides followed the Freundlich model. The XAD-16 resin was the most effective resin for the enrichment of peptides due to its high adsorption and total desorption capacities. Interestingly, the umami peptides were enriched in the deionized water fraction. This study provides new insights into exploring performance and separation characteristics of macroporous resins on soy sauce, and indicated that peptide may be the contributor to the umami taste in Chinese soy sauce., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study focused on the enzymatic characteristics, with regard to primary specificity, secondary specificity, and hydrolysis ability of five proteases (Alcalase, Protamex, papain, Flavourzyme, and ProtexA), towards the release of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from bovine casein. The secondary specificity of the five proteases, especially the release capability for Xaa-Pro-type peptide played a determining role in the release of DPP-IV inhibitory peptides from bovine casein. Moreover, protease combinations, chosen based on their Xaa-Pro-type peptide release abilities, resulted in superior DPP-IV inhibitory activity compared to other combinations. Particularly, the hydrolysate with the highest DPP-IV inhibitory activity (IC 50 value of 0.45 ± 0.07 mg/mL) was obtained by the combination hydrolysis of ProtexA and papain, achieving a 74.63% ± 1.62% (peak area) release of Xaa-Pro-type peptide, and a significant improving glucose tolerance in mice (33.42% ± 1.68% reduction of blood glucose AUC compared with control mice). The mechanistic insights revealed that papain efficiently released long Xaa-Pro-type peptides in the early hydrolysis stage, and subsequently, the synergistic effect of papain and ProtexA led to an accelerated and extensive release of short Xaa-Pro-type peptides. These findings provide valuable insight into enzymatic processes for generating DPP-IV inhibiting peptides from casein. [Display omitted] • The enzymatic characteristics of Alcalase, Protamex, papain, Flavourzyme, and ProtexA on the release of DPP-IV inhibitory peptides from casein were studied. • Secondary specificity of proteases determined the release of DPP-IV inhibitory peptides. • Protease combinations, based on their Xaa-Pro-type peptide release abilities, could release more DPP-IV inhibitory peptides. • Casein hydrolyzed by the combination of ProtexA and papain had a 74.63% ± 1.62% release of Xaa-Pro-type peptide. [ABSTRACT FROM AUTHOR]

The search for treatment of aging and age-related diseases is a public health issue. Sea cucumber ( Cucumaria frondosa ) hydrolysate (CFH) prepared by enzymatic hydrolysis and purified by ultrafiltration was examined for its peptide profile and ability to prevent and/or delay aging in fruit flies and age-related neurodegenerative disorders in d -galactose-induced aging mice. CFH contained peptides with low molecular weights (mainly < 3 kDa), Ala, Val and Leu at the N-terminal end and aromatic/hydrophobic/acidic amino acids in sequence. We provided the first in vivo evidence that CFH significantly prolonged the lifespan of fruit flies and ameliorated learning and memory deficits of d -galactose-induced aging mice. The mechanism underlying its protective effect might be associated with the ability to up-regulate Klotho expression, increase the activities of SOD and GSH-Px, inhibit lipid peroxidation and protein oxidation, and down-regulate acetylcholinesterase activity. CFH could be a health-promoting ingredient in coping with aging and age-related diseases. [ABSTRACT FROM AUTHOR]

DPPH assay is widely used to evaluate the radical scavenging activities of peptides. Effects of pH and buffers on the stability of DPPH• and its reduced product (DPPHH) in the ethanol-buffer solution were investigated in this study and the reactivity of DPPH• towards several dipeptides was compared to that of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS• + ) and the peroxyl radicals in oxygen radical absorbance capacity (ORAC) assay. Results showed that the deprotonation of DPPHH under basic condition could interfere with the spectrophotometric measurement at 515–525 nm. It was suggested that the reaction mixture be maintained at a final pH range of 5.0–6.5 in 1:1 ethanol–acetate/citrate buffer medium when evaluating the activities of peptides. Additionally, among tested dipeptides, only Cys-containing dipeptides displayed DPPH• scavenging activity with 0.14–0.28 μmol TE (Trolox equivalent)/μmol, while Tyr/Trp-containing dipeptides with high reactivity towards ABTS• + and peroxyl radicals were inert to DPPH• with TE values less than 0.02. [ABSTRACT FROM AUTHOR]

Moringa oleifera Lam. leaf (MOL), a rich source of protein and phenolics, was traditionally used to treat various diseases including headaches, fevers, sore throat and dyslipidemia. Recently, MOL was reported to possess antioxidant, anti-dyslipidemia and hepato-renal protective activities, indicating that MOL could become a potential agent to improve metabolic disorders associated with hyperuricemia. The antihyperuricemic effect of MOL hydrolysate (MOLH) with high contents of phenolics and peptides remains unknown. The aim of this study is to investigate xanthine oxidase (XO) inhibitory activity of MOLH, to clarify phenolic and peptide profiles of MOLH, and to evaluate possible mechanism underlying the antihyperuricemic effect of MOLH. MOLH was prepared by enzymatic hydrolysis using commercial trypsin. XO inhibitory activity was determined by XO reaction-UPLC-MS coupling method. The chemical profiles of the phenolic and peptide fractions of MOLH were determined by UPLC-QTOF-MS/MS. The antihyperuricemic effect of MOLH was evaluated in a potassium oxonate-induced hyperuricemic rat model at doses of 200 and 500 mg/kg. Serum uric acid (UA), urea nitrogen, creatinine (CRE), triglyceride (TG), total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels, serum XO activity, liver malondialdehyde (MDA) equivalent level, renal tumor necrosis factor-α and interleukin-1β levels, and protein expression of renal urate-anion transporter 1, glucose transporter 9 and ATP-binding cassette transporter G2 were determined. The phenolic and peptide fractions played key roles in inhibiting XO activity and blocking uric acid production. Five flavonoids and sixteen polypeptides were identified in the phenolic and peptide fractions of MOLH, respectively. MOLH (200 and 500 mg/kg) could effectively reduce the serum UA level of hyperuricemic rats (p < 0.001) by regulation of serum XO activity (p < 0.05 at 200 mg/kg, p < 0.01 at 500 mg/kg) and renal urate transporters. Besides, MOLH could improve metabolic disorders associated with hyperuricemia by its multiple actions on liver MDA (p < 0.001), serum CRE (p < 0.05 at 500 mg/kg) and serum TG (p < 0.001). The results provided scientific evidence that MOLH rich in phenolics and peptides ameliorated hyperuricemia and metabolic disorders. This study validated the potential use of MOLH for regulation of hyperuricemia. Image 1 • Moringa oleifera leaf hydrolysate exhibited significant antihyperuricemic effect. • M. oleifera leaf flavonoids and peptides contributed to antihyperuricemic effect. • M. oleifera leaf hydrolysate regulated xanthine oxidase and urate transporters. • M. oleifera leaf hydrolysate improved hyperuricemia and metabolic disorders. [ABSTRACT FROM AUTHOR]

Defatted walnut meal (DWM), a main byproduct of walnut oil production, is rich in proteins with a high essential amino acid content. Our research was focused on the functional activities of defatted walnut meal hydrolysate (DWMH). DWMH exhibited relatively strong hydroxyl scavenging and oxygen radical absorbance capacities, protective effect on H 2 O 2 -injured PC12 cells compared with GSH and cerebrolysin. Besides, DWMH could combat d -galactose induced learning and memory impairments in the Morris water maze test and in the Dark/light avoidance test of mice. Seventy-seven peptides were identified by UPLC-ESI-MS/MS in the most potent antioxidative fraction of DWMH. WSREEQEREE and ADIYTEEAGR were the peptides that most likely accounted for the suppression of H 2 O 2 -induced apoptosis in PC12 cells. The mixture of the commercially available amino acids did not offer any protection on H 2 O 2 -injured PC12 cells, suggesting that amino acid sequence rather than amino acid composition was the dominant influencing factor. DWMH could be recommended as natural antioxidants for the development of functional foods targeting memory impairments. [ABSTRACT FROM AUTHOR]

Highlights • Identification of six novel umami and umami-enhancing peptides from PPIH. • RGENESEEEGAIVT was the first discovered umami and umami-enhancing tetradecapeptide. • Peptides could enhance the overall taste of chicken soup and soy sauce. • E-tongue has the potential to distinguish taste attributes of peptide solutions. Abstract Six novel peptides were separated from peanut protein isolate hydrolysate (PPIH) using ethanol precipitation and gel chromatography, and identified as DQR, NNP, EGF, EDG, TESSSE and RGENESEEEGAIVT by UPLC–ESI–QTOF–MS/MS. On the basis of sensory results, all peptides were perceived umami with threshold values from 0.39 to 1.11 mM and had umami-enhancing abilities simultaneously with threshold values from 0.33 to 0.82 mM. RGENESEEEGAIVT was the first discovered tetradecapeptide with umami and umami-enhancing ability. The dose–response test revealed that umami-enhancing activities of identified peptides were different: TESSSE and RGENESEEEGAIVT imparted better umami intensity when equimolar monosodium glutamate (MSG) was added into 0.5 g/L MSG solution. Taste profile analyses of complex mixtures with/without synthetic peptides were determined by both electronic tongue and human panellists, suggesting that umami peptides influence multiple tastes and electronic tongue has the potential to replace sensory test to distinguish taste attributes of foods rich in peptides. [ABSTRACT FROM AUTHOR]