Pea Protein Hydrolysate Research Articles

Targeted discovery of pea protein-derived GLP-1-secreting peptides by CaSR activation-based molecular docking and their digestive stability

Dietary proteins could stimulate Glucagon-like peptide 1 (GLP-1) secretion. However, only a few food-derived GLP-1-secreting peptides have been identified. Herein, three GLP-1-secreting peptides were identified from pea protein hydrolysate (PPH) by calcium-sensing receptor (CaSR) activation-based molecular docking. PPH-triggered GLP-1 secretion was mediated by CaSR activation. A total of 4221 peptides were sequenced from PPH through peptidomic analysis. Subsequently, three GLP-1-secreting peptides, including RFY, FEPF, and FLFK, were screened by CaSR activation-based molecular docking, and peptide-induced GLP-1 secretion were mediated by CaSR activation. More importantly, FEPF and FLFK exhibited good digestive stability. The molecular docking suggested that binding energy between peptides and CaSR was negatively correlated with their ability to stimulate GLP-1 secretion, and some binding sites in CaSR, such as Asn102 and Tyr218, play a crucial role in stimulating GLP-1 secretion. Our findings suggest that the targeted discovery of pea protein-derived GLP-1-secreting peptides through CaSR activation-based molecular docking is an effective strategy.

Yogurt fortified with various protein hydrolysates: Texture and functional properties

This work evaluated the impact of incorporating 1% of commercial protein hydrolysates [rice protein hydrolysate (RPH), pea protein hydrolysate (PPH), and casein hydrolysate (CH)] on the functional, microstructure, and texture properties of set yogurt. Yogurt prepared with RPH exhibited the highest viability number of Streptococcus thermophilus. The addition of three hydrolysate types to yogurt revealed significant increases in the antioxidant and ACE-inhibitory activities, where the highest values were noted for the yogurt prepared with RPH. RPH exhibited no differences in texture properties (firmness, consistency, and cohesiveness) to control yogurt. These results were confirmed by scanning electron microscope examination. RPH and control yogurts showed compacted and dense structures accompanied by small pores, whereas CH and PPH yogurt structures were characterized by coarse networks with large voids. Furthermore, there was no significant impact of adding protein hydrolysates on the overall acceptability of yogurt as indicated by a sensory panel.

Characterisation of functional pea protein hydrolysates and their immunomodulatory activity

SummaryPea proteins has been widely used in food industry due to its functionality and health benefits. Pea peptides derived from pea proteins are considered as promising source of novel functional food. However, the chemical structure of pea peptides with immunomodulatory activity remains unclear. In this work, 46 pea peptides with molecular weight from 533.28 to 1462.78 were identified from pea protein hydrolysates. The hydrolysates could significantly increase the immunomodulatory activity of the macrophages by elevation of phagocytic activity, promoting the production of nitric oxide and pro‐inflammatory cytokines (TNF‐α and IL‐6) at the doses of 0.25–1.0 mg mL−1. Moreover, upregulation of iNOS, TNF‐α and IL‐6 were related to the activity of pea protein hydrolysates. Fourteen peptides with activity scores > 0.3 showed good affinity with TLR4/MD‐2 by molecular docking. TLR4 activation was involved in the immunomodulatory activity of bioactive pea peptides through a hydrogen bond, Pi‐cation, Pi‐Sigma, Pi‐Pi stacked and/or Pi‐Pi T‐shaped interactions respectively. The above results indicated that pea peptides could be used as healthy food with immunomodulatory function, promoting the development of pea proteins in the functional food industry.

Pea protein hydrolysate stimulates GLP-1 secretion in NCI-H716 cells via simultaneously activating the sensing receptors CaSR and PepT1.

Glucagon-like peptide-1 (GLP-1) plays a crucial role in regulating glucose homeostasis by stimulating insulin secretion and suppressing glucagon release. Our previous study observed that pea protein hydrolysate (PPH) exhibited the function of triggering GLP-1 secretion. However, the underlying mechanisms have not been revealed. Herein, the mechanisms of PPH-stimulated GLP-1 secretion were investigated in NCI-H716 cells. The PPH-induced GLP-1 secretion was reduced (p < 0.05) after adding the sensing receptor antagonists NPS-2143 and 4-AMBA, indicating that activation of both calcium-sensing receptor (CaSR) and peptide-transporter 1 (PepT1) was involved in PPH-triggered GLP-1 release. Moreover, the intracellular Ca2+ level increased by 2.01 times during the PPH-induced GLP-1 secretion. Similarly, the cAMP content also increased by 1.43 times after stimulation by PPH. The RT-qPCR results showed that PPH increased the gene expression of prohormone convertase 1/3 (PCSK-1) by 2.79-fold, which effectively promoted the conversion of proglucagon (GCG) to GLP-1. The specific pathway of PPH-induced GLP-1 secretion may involve both CaSR and PepT1 activation-induced Ca2+ influx and cAMP generation, which effectively enhanced the enzyme activity of prohormone convertase 1/3 (PCSK-1) and ultimately promoted GLP-1 secretion.

Pea protein hydrolysate reduces blood glucose in high-fat diet and streptozotocin-induced diabetic mice.

Food proteins have been recognized as an ideal source to release bioactive peptides with the potential to intervene nutrition related chronic diseases, such as cardiovascular diseases, obesity and diabetes. Our previous studies showed that pea protein hydrolysate (PPH) could suppress hepatic glucose production in hepatic cells via inhibiting the gluconeogenic signaling. Thus, we hypothesized that PPH could play the hypoglycemic role in vivo. In the present study, the mice model with type 2 diabetic mellitus (T2DM) was developed by high-fat diet and low dose of streptozotocin injections. PPH was administered orally with a dosage of 1000 mg/kg body weight for 9 weeks, followed by the downstream biomedical analyses. The results showed that the 9-week treatment of PPH could reduce fasting blood glucose by 29.6% and improve glucose tolerance in the T2DM mice. The associated mechanisms included suppression of the gluconeogenic pathway, activation of the insulin signaling and modulation of the renin angiotensin system in the liver of the diabetic mice. In addition, the levels of pro-inflammatory markers in both liver and serum were reduced by the PPH treatment. The hypoglycemic effect of PPH in T2DM mice was demonstrated in the present study. Findings from this study could provide rationale to incorporate PPH into functional foods or nutraceuticals for glycemic control.

Development of low‐fat Mozzarella cheeses enriched with soy or pea protein hydrolysates: Composition, texture and functional properties during ageing

To explore the effect of plant protein hydrolysate (PPHS, soy and pea protein hydrolysate) on the characteristics of low‐fat Mozzarella cheeses (LFCs), samples incorporating PPHS were prepared and analysed for pizza bake performance, texture, meltability, sensory attributes and microstructure. Upon addition of an appropriate amount of PPHS to cheese milk, the pizza bake performance, stretch performance and sensory properties of LFC were improved, while the texture values (hardness, stickiness and stretch force) decreased gradually. Cheese matrix exhibited an irregular honeycomb structure as the PPHS content increased. Therefore, PPHS holds promise as substitute for milk protein, and LFC containing PPHS represents a highly promising low‐fat dairy product with improved quality and diversified ingredients.

The Umami-enhancing Ability Improvement of Pea Protein Hydrolysate by Maillard Reaction Utilizing Sugar-derived NADES

The Umami-enhancing Ability Improvement of Pea Protein Hydrolysate by Maillard Reaction Utilizing Sugar-derived NADES

Potential antidiabetic effect of muffins formulated with different protein hydrolysates: Role of bioactive peptides formed during digestion

This study aimed to evaluate whether the reformulation of a conventional muffin (CM) by incorporating hydrolysates of casein, soybean protein, pea protein, and rice protein at two concentrations as wheat flour replacement, affected the glycemic index (GI) and the release of bioactive peptides (BAPs) during the digestion in vitro.Compared to CM, muffins with hydrolyzed pea and rice proteins lowered GI by 15% and 5%, respectively, without a significant dose-dependent effect; moreover, during digestion they delivered BAPs, including some amylase-inhibitors. Conversely, the muffins incorporating casein hydrolysate at high concentration showed the highest GI and delivered highest amount of opioid and DPPIV-inhibitor BAPs. Soy protein hydrolysates did not significantly affect the GI or BAPs released.Findings suggested that reformulating a muffin with protein hydrolysates, besides a reduced GI, as in the case of pea and rice protein hydrolysates, may provide further biological effects through the release of BAPs during the digestion.

Softening fat-free cream cheese by incorporating aggregates of pea protein hydrolysates

Protein-based fat replacers are increasing in popularity due to their low-calorie nature and customers' preference on high protein foods. Nevertheless, their fabrication requires large energy input (75–95 °C, 20–40 min) which limits their food application. In the present study, pea protein isolates (PPI) were hydrolyzed by Alcalase, followed by mild heating (85 °C for 10 min) to form aggregates as fat replacer. Protein aggregates were added to skim milk at various concentrations (0.1, 0.3, 0.5%) to develop fat-free cream cheese. Chemical analysis found incorporation of pea protein hydrolysates aggregates (PPH) into fat-free cream cheese increased the moisture content from ∼63% to ∼70%. Rheological measurements showed the storage modulus (G’) was decreased from 36 kPa in the control cheese to 11 kPa and 25 kPa when at 0.3% PPH and PPI aggregates were added. Apparent viscosity (η) was decreased ∼78% and ∼36% with the addition of 0.3% PPH and PPI, respectively. Creep-recovery test showed under the same stress, the strain (%) and α, λ1, λ2 of cream cheese containing PPH had much larger value than those of PPI. The Confocal and Scanning Electron Microscopy showed a less continuous network with large aggregates in PPH contained cream cheese compared to those of PPI or negative control sample, possibly due to clustering of PPH or PPH-casein complex. The information provided from this study sheds light on the development of desirable plant protein-based fat replacer with higher energy efficiency for potential application of low-fat dairy foods.

Evaluating the In Situ Insulinotropic Effects of Pea Protein Hydrolysates Mediated by Active GLP-1 via a 2D and Dual-Layered Coculture Cell Model.

The aim of this study was to evaluate the in situ insulinotropic effects of pea protein hydrolysates (PPHs) mediated by active glucagon-like peptide-17-36 (active GLP-1) using a 2D and dual-layered coculture cell model. Following this model, a mixed Caco-2 and NCI-H716 cell monolayer was differentiated on the apical side to study the effects of PPHs on active GLP-1 levels; meanwhile, the beta-TC-6 cells were seeded on the basolateral side to investigate the insulin responses induced by active GLP-1. The in situ DPP-4 half-maximal inhibitory concentration (IC50) of PPHs, PPHs-120G, and PPHs-120I was 2.94, 3.43, and 2.26 mg/mL, respectively. They directly stimulated active GLP-1 secretion in NCI-H716 cells by 3.03 ± 0.21, 1.99 ± 0.03, and 2.24 ± 0.02 times, respectively. Insulin release in beta-TC-6 cells was directly stimulated by PPHs but not by PPHs-120G and PPHs-120I. Interestingly, PPHs-120G and PPHs-120I indirectly stimulated insulin release in this coculture cell model by enhancing active GLP-1 concentrations. More importantly, PPHs, PPHs-120G, and PPHs-120I increase active GLP-1 levels by their dual function of stimulating active GLP-1 secretion and DPP-4 inhibition. This study suggests that the 2D and dual-layered coculture cell model supports a more comprehensive assessment of in situ insulinotropic effects of protein hydrolysates mediated by active GLP-1.

Neuroprotective peptides isolated from flavourzyme-pea protein hydrolysate protect human SH-SY5Y cells from Aβ1-42 induced apoptosis

Neuroprotective peptides derived from Flavourzyme®-pea protein hydrolysate (FPPH) were sequentially purified by chromatographies. Cell viability of β-amyloid peptide-induced oxidative damage in human neuroblastoma SH-SY5Y cells was used to explore neuroprotective effects of FPPH. Three novel neuroprotective peptides were obtained by liquid chromatography tandem mass spectrometry. In vitro effect of gastrointestinal proteases on neuroprotective effects of the three peptides was also investigated. The result suggested that the gastrointestinal proteases did not affect neuroprotective effects of the three novel peptides, which reveals the potential to ameliorate diseases caused by neurodegeneration. Pretreating SH-SY5Y cells with peptide GGPFKSPF (GF) before Aβ1-42 treatment increased cell viability and reduced formation of reactive oxygen species. Decrease of mitochondrial membrane potential and anti-apoptotic proteins Bcl-2 induced by Aβ1-42 can be restored by GF treatment. GF can remarkably decrease the contents of pro-apoptotic proteins, Bax and Caspase-3, inducing the activation of Akt/GSK-3β pathway by phosphorylation of Akt. GF exerted an antioxidant effect via Nrf2/HO-1 signaling pathway by activating Nrf2, reducing malondialdehyde level as well as increasing the expression of HO-1 and the activities of superoxide dismutase, catalase and glutathione peroxidase. As a results, GF had preventively potential as functional foods in Aβ–related neurodegenerative disease.

Improved printability of pea protein hydrolysates for protein-enriched 3D printed foods

Improved printability of pea protein hydrolysates for protein-enriched 3D printed foods

Maillard reaction products of pea protein hydrolysate as a flavour enhancer for beef flavors: Effects on flavor and physicochemical properties.

This study evaluated the effects of Maillard reaction products of pea protein hydrolyzates (MRPs-PPH) as salt-reducing and umami-enhancing components on the flavor and physicochemical properties of beef flavors. The addition of MRPs-PPH reduced the brightness of beef flavors, increased the redness and yellowness, as well as changed the texture characteristics of beef flavors. With the addition of MRPs-PPH, the apparent viscosity, storage modulus and loss modulus of beef flavors decreased. Finally, the relationship between taste attributes and flavor compounds of the samples was analyzed by Partial Least Squares Regression (PLSR), and flavor compounds with significant positive contributions to different taste attributes were found. This study showed that MRPs-PPH could be used as a flavor enhancer derived from biomacromolecules with salt reduction and freshness enhancement.

Synthesis of taste active γ-glutamyl peptides with pea protein hydrolysate and their taste mechanism via in silico study

Pea (Pisum sativum L.) protein hydrolysate (PPH) has a bitter taste, which has limited its use in food industry. γ-Glutamylation is used to debitter PPH. Results showed that the bitterness of PPH was decreased significantly due to the formation of γ-glutamyl peptides, including 16 γ-[Glu](n=1/2)-amino acids (AAs) and 8 newly discovered γ-glutamyl tripeptides (γ-Glu-Asn-Phe, γ-Glu-Leu-Val, γ-Glu-Leu-Tyr, γ-Glu-Gly-Leu, γ-Glu-Gly-Phe, γ-Glu-Gly-Tyr, γ-Glu-Val-Val, and γ-Glu-Gln-Tyr). Their total production concentrations were 27.25 μmol/L and 77.76 μmol/L, respectively. The γ-Glu-AA-AAs presented an umami-enhancing, salty-enhancing, and kokumi taste when their concentration reached 1.67 ± 0.20 ∼ 2.07 ± 0.20, 1.65 ± 0.25 ∼ 2.29 ± 0.45 and 0.68 ± 0.19 ∼ 1.03 ± 0.22 mmol/L, respectively. The γ-Glu-AA-AAs exhibited a kokumi taste by entering the Venus flytrap (VFT) of the calcium-sensing receptor and interacting with Ser147, Ala168, and Ser170. γ-Glu-AA-AAs can enhance the umaminess of Monosodium Glutamate (MSG) as they can enter the binding pocket of the taste receptor type 1 subunit 3 (T1R3)-MSG complex.

Investigating structure, biological activity, peptide composition and emulsifying properties of pea protein hydrolysates obtained by cell envelope proteinase from Lactobacillus delbrueckii subsp. bulgaricus

We present the structure, biological activity, peptide composition, and emulsifying properties of pea protein isolate (PPI) after hydrolysis by cell envelope proteinase (CEP) from Lactobacillus delbrueckii subsp. bulgaricus. Hydrolysis resulted in the unfolding of the PPI structure, characterized by an increase in fluorescence and UV absorption, which was related to thermal stability as demonstrated by a significant increase in ΔH and the thermal denaturation temperature (from 77.25 ± 0.05 to 84.45 ± 0.04 °C). The hydrophobic amino acid of PPI significantly increased from 218.26 ± 0.04 to 620.77 ± 0.04 followed by 557.18 ± 0.05 mg/100 g, which was related to their emulsifying properties, with the maximum emulsifying activity index (88.62 ± 0.83 m2/g, after 6 h hydrolysis) and emulsifying stability index (130.77 ± 1.12 min, after 2 h hydrolysis). Further, the results of LC-MS/MS analysis demonstrated that the CEP tended to hydrolyze peptides with an N-terminus dominated by Ser and a C-terminus dominated by Leu, which enhanced the biological activity of pea protein hydrolysates, as supported by their relatively high antioxidant (ABTS+ and DPPH radical scavenging rates were 82.31 ± 0.32% and 88.95 ± 0.31%) and ACE inhibitory (83.56 ± 1.70%) activities after 6 h of hydrolysis. 15 peptide sequences (score > 0.5) possessed both antioxidant and ACE inhibitory activity potential according to the BIOPEP database. This study provides theoretical guidance for the development of CEP-hydrolyzed peptides with antioxidant and ACE inhibitory activity that can be used as emulsifiers in functional foods.

Purification, Identification and Evaluation of Antioxidant Peptides from Pea Protein Hydrolysates.

Food-derived antioxidant peptides can be explored as natural antioxidants due to their potential health benefits. In this study, antioxidant peptides were isolated and purified from pea protein hydrolysates (PPH). The DPPH and ABTS radical scavenging activities were used as indexes to purify the antioxidant peptides by a series of purification steps including ultrafiltration, ion exchange chromatography, G25 gel filtration chromatography, and reversed-phase chromatography. Three novel antioxidant peptides YLVN, EEHLCFR and TFY were identified, which all exhibited strong antioxidant activity in vitro. EEHLCFR showed stronger DPPH scavenging activity with an IC50 value of 0.027 mg/mL. YLVN showed stronger ABTS scavenging activity with an IC50 value of 0.002 mg/mL and higher ORAC values of 1.120 ± 0.231 μmol TE/μmol, which is even better than that of GSH. Three novel antioxidant peptides significantly elevated LO2 cells viability even at the concentration of 0.025 mg/mL, and cell viability enhanced to 53.42 ± 1.19%, 55.78 ± 1.03%, and 51.09 ± 1.06% respectively, compared to that of H2O2 injury group (48.35 ± 0.96%), and prevented the accumulation of ROS by enhancing the activities of antioxidant enzymes in H2O2-induced oxidative stress LO2 cells. The molecular docking results showed that the potential molecular mechanism of the three novel antioxidant peptides may be in high correlation with the activation of the Keap1-Nrf2 pathway by occupying the Keap1-Nrf2 binding site. These results demonstrate that the three novel antioxidant peptides are potential natural antioxidants that can be devoted to medicine or functional food ingredients.

Interfacial Properties of Pea Protein Hydrolysate: The Effect of Ionic Strength

The effect of a tryptic hydrolysis as well as the effect of ionic strength (0–0.4 M NaCl) was investigated on the oil/water interfacial properties of soluble pea protein hydrolysate (SPPH) at neutral pH and room temperature (20 ± 0.01 °C). SEC-MALS and SDS-Page analysis showed that tryptic hydrolysis created a lower molecular weight polypeptide mixture, whereas FTIR analysis and DSC thermograms demonstrated a more disordered and flexible structure. The bulk properties of SPPH were studied in terms of hydrodynamic diameter and turbidity, where higher particle size (+ ~13 nm) and turbidity were observed at 0.4 M NaCl. Regarding the interfacial properties, the surface activity of SPPH improved by increasing ionic strength, with maximum interfacial pressure (14.28 mN/m) at 0.4 M NaCl. Nevertheless, the addition of NaCl negatively affected the elasticity and strength of the interfacial film, where the sample without salt exhibited the highest dilatational and shear storage modulus in all the frequencies considered.

Metal-chelating activity of soy and pea protein hydrolysates obtained after different enzymatic treatments from protein isolates

Soy and pea proteins are two rich sources of essential amino acids. The hydrolysis of these proteins reveals functional and bioactive properties of the produced small peptide mixtures. In our study, we employed the hydrolysis of soy and pea protein isolates with the endopeptidases Alcalase® and Protamex®, used alone or followed by the exopeptidase Flavourzyme®. The sequential enzyme treatments were the most efficient regarding the degree of hydrolysis. Then, soy and pea protein hydrolysates (SPHs and PPHs, respectively) were ultrafiltrated in order to select peptides of molecular weight ≤ 1 kDa. Whatever the protein source or the hydrolysis treatment, the hydrolysates showed similar molecular weight distributions and amino acid compositions. In addition, all the ultrafiltrated hydrolysates possess metal-chelating activities, as determined by UV-spectrophotometry and Surface Plasmon Resonance (SPR). However, the SPR data revealed better chelating affinities in SPHs and PPHs when produced by sequential enzymatic treatment.

In vitro inhibition of acetylcholinesterase activity by yellow field pea (Pisum sativum) protein-derived peptides as revealed by kinetics and molecular docking.

Compounds with structural similarities to the neurotransmitter (acetylcholine) are mostly used to inhibit the activity of acetylcholinesterase (AChE) in Alzheimer's disease (AD) therapy. However, the existing drugs only alleviate symptoms of moderate to mild conditions and come with side effects; hence, the search is still on for potent and safer options. In this study, High performance liquid chromatography (HPLC) fractionations of AChE-inhibitory pea protein hydrolysates obtained from alcalase, flavourzyme and pepsin digestions were carried out followed by sequence identification of the most active fractions using mass spectrometry. Subsequently, 20 novel peptide sequences identified from the active fractions were synthesized and five peptides, QSQS, LQHNA, SQSRS, ETRSQ, PQDER (IC50 = 1.53 - 1.61 μg/mL) were selected and analyzed for ability to change AChE protein conformation (fluorescence emission and circular dichroism), kinetics of enzyme inhibition, and enzyme-ligand binding configurations using molecular docking. The kinetics studies revealed different inhibition modes by the peptides with relatively low (<0.02 mM and <0.1 mM) inhibition constant and Michaelis constant, respectively, while maximum velocity was reduced. Conformational changes were confirmed by losses in fluorescence intensity and reduced α-helix content of AChE after interactions with different peptides. Molecular docking revealed binding of the peptides to both the catalytic anionic site and the peripheral anionic site. The five analyzed peptides all contained glutamine (Q) but sequences with Q in the penultimate N-terminal position (LQHNA, SQSRS, and PQDER) had stronger binding affinity. Results from the different analysis in this study confirm that the peptides obtained from enzymatic digestion of pea protein possess the potential to be used as novel AChE-inhibitory agents in AD management.

Pea Protein-Derived Peptides Inhibit Hepatic Glucose Production via the Gluconeogenic Signaling in the AML-12 Cells.

Pea protein is considered to be a high quality dietary protein source, but also it is an ideal raw material for the production of bioactive peptides. Although the hypoglycemic effect of pea protein hydrolysate (PPH) has been previously reported, the underlying mechanisms, in particular its effect on the hepatic gluconeogenesis, remain to be elucidated. In the present study, we found that PPH suppressed glucose production in mouse liver cell-line AML-12 cells. Although both of the gluconeogenic and insulin signaling pathways in the AML-12 cells could be regulated by PPH, the suppression of glucose production was dependent on the inhibition of the cAMP response element-binding protein (CREB)-mediated signaling in the gluconeogenic pathway, but not the activation of insulin signaling. Findings from the present study have unveiled a novel role of PPH underlying its anti-diabetic activity, which could be helpful to accelerate the development of functional foods and nutraceuticals using PPH as a starting material.