Pea Albumins Research Articles

A time-resolved investigation at multiple-length scales of the structure of liquid foam stabilized by albumins from pea

HypothesisThe structural details of foams made with pea albumins are affected by the pH of the initial solution and followed heat treatment. ExperimentsAn in situ, time-resolved investigation of foams prepared with pea albumins was conducted using small-angle neutron scattering (SANS) in combination with imaging and conductance measurements. Solutions were tested at pH three pH values (3, 4.5, and 8) before and after heating (90 °C for 1 and 5 min). FindingsThe characteristic structures present in the foam from the nano to the meso-scale differed during drainage depending on solution pH. Foams obtained at pH 3, had the largest bubble radius and thinnest plateau border, as well as the highest extent of liquid drainage. At pH 4.5, close to the isoelectric point of the proteins, foams displayed similar bubbles’ behavior to those at pH 8, but with the largest film thickness. In this case, the proteins were extensively aggregated. Heating of the solutions prior to foaming did not significantly affect the foam aging regardless of pH. The quantification of specific surface areas and film thickness over time without sample disruption shows to be a powerful approach to designing foam structures.

Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

Protein composition and nutritional aspects of pea protein fractions obtained by a modified isoelectric precipitation method using fermentation.

Pea albumins are promising for their nutritional, biological, and techno-functional properties. However, this fraction is usually discarded in the industry due to its low protein content compared to globulin fraction and the presence of some anti-nutritional compounds. In the present study, we used an alternative method of pea protein extraction based on alkaline solubilization/isoelectric precipitation in which the reduction of pH was achieved by lactic acid fermentation using specific starters instead of mineral acids. Hence, the main objective of this study was to examine the protein profile and the content of anti-nutritional and nutritional active compounds in pea albumin-rich fractions obtained by the isoelectric extraction method without (control) or with fermentation with different lactic acid bacteria (Streptococcus thermophilus, Lactiplantibacillus plantarum, and their co-culture). Different pea cultivars (Cartouche, Ascension, and Assas) were used here for their differences in protein profile. The results revealed a higher total nitrogen content in albumin-rich fraction for fermented samples and, in particular, for co-culture. The majority of total nitrogen was determined as non-protein (~50%), suggesting the degradation of proteins by LAB to small peptides and amino acids, which were solubilized in the soluble fraction (albumin) as confirmed by size exclusion chromatography (SEC-HPLC) analysis. Moreover, the higher antioxidant activity of fermented albumin samples was attributed to the production of small peptides during extraction. Lactic acid fermentation also resulted in a significant reduction of trypsin inhibitor activity, α-galactoside, and phytic acid content of this fraction compared to control.

Recovery and Utilization of Pea Albumins as Acidic Emulsion Stabilizer by Complexation with Dextran Sulfate.

In this work, pea albumins (PAs) were efficiently recovered by complexation with dextran sulfate (DS), and the emulsifying ability and stability of PA/DS complexes were studied. The largest amounts of PAs (81.25%) were recovered at r = 5:1 and pHmax (pH 3.41) by forming insoluble complexes; and only soluble complexes were formed at r = 2:1 and over the whole pH range (2.0-7.0). The emulsions stabilized by PA/DS soluble complexes remained stable under acidic conditions due to the highly negatively charge (from -45.10 ± 0.40 to -57.23 ± 0.66 mV) and small particle size (0.168 ± 0.010-0.448 ± 0.004 μm), while emulsions stabilized by PAs alone generated a strong creaming and serum separation at pH 5 and 6. In terms of emulsifying stability, all PA emulsions and unheated PA/DS emulsions became unstable with different creaming index after 14 days storage. SDS-PAGE results showed that the interface adsorption proteins of unheated emulsions mainly consisted of PA1a, which was unfavorable to the stability of the interface. On the contrary, heat treatment (95 °C, 30 min) and complexation (PA/DS = 2:1) enhanced the adsorption of PA2 and lectin at the interface, inhibiting the aggregation of PA2 and lectin. This resulted in long-term stability of the PA/DS emulsions under acidic conditions.

Coacervation in pea protein solutions: The effect of pH, salt, and fractionation processing steps

Legumes such as pea and soy have the ability to form coacervates in solution, which depends on the pH and salt concentration. We studied the phenomenon of coacervation for pea protein solutions in which the proteins were exposed to different extents of fractionation (i.e., neutral extraction, neutral extraction followed by freeze drying, alkaline extraction and alkaline extraction followed by isoelectric precipitation). The pH of the dispersed fractions was varied between 5.5 and 6.5 and the salt concentration between 0 and 500 mM NaCl. Confocal microscopy and particle size measurements were used to confirm the presence of spherical shaped protein-rich domains; a signature of the concentrated coacervate phase. It was found that the mildest processed fraction – obtained after protein extraction from the flour and subsequent removal of solids by centrifugation – formed coacervates as well as aggregates (i.e., non-spherical domains), between pH 6.0 and 6.5. At pH 6.25 only coacervates were observed. When 50 mM NaCl was added at pH 6.25, the coacervate average diameter increased from approximately 1 to 5 μm. When the salt concentration was increased to ≥200 mM NaCl, no coacervates were observed anymore. The coacervates formed at pH 6.25 with 0 and 50 mM NaCl were examined further. It turned out that the coacervates contained pea globulins, with legumin being most abundant. Pea albumins were not found in the coacervates. The internal protein content of the coacervates formed at pH 6.25 was around 45 wt %. When the mildest processed fraction was freeze dried, coacervates could still be formed at pH 6.25 and low salt concentrations (≤50 mM NaCl). After alkaline extraction, dispersions with both aggregates and coacervates were observed at pH 6.25. Isoelectric precipitated pea protein isolate did not show coacervates at any of the conditions tested. Our research shows that mildly fractionated pea protein was most suitable to form coacervates.

Complexation of pea albumins with anionic polysaccharides and purification of PA1a

Selective complexation with anionic polysaccharides (sulfated (SPS) and carboxylated polysaccharides (CPS)) was used to purify a target protein, pea albumin 1a (PA1a), a sulfur rich protein, from PA1a/pea albumin 2 (PA2) mixtures (PPM, two main pea albumins). The results showed that PPM-SPS system had higher critical pHc and protein recovery than that of PPM-CPS. The binding affinity of PA2 with CPS or SPS was stronger than that of PA1a, which led to the preferential complexation of PA2 and the enrichment of single PA1a in the supernatant. The high charge densities of dextran sulfate, κ-carrageenan and sodium alginate were unfavorable to the isolation of PA1a, and the highest PA1a recovery was obtained for PPM-carboxymethyl cellulose system, although it had the lowest charge density. After removal of polysaccharides by ultrafiltration in non-interacting pH conditions, a purified PA1a (>90% by SEC-HPLC) was obtained. This work provides a novel strategy for the purification of PA1a from multi-proteins system based on selective complexation.

A Pea (Pisum sativum L.) Seed Vicilins Hydrolysate Exhibits PPARγ Ligand Activity and Modulates Adipocyte Differentiation in a 3T3-L1 Cell Culture Model.

Legume consumption has been reported to induce beneficial effects on obesity-associated metabolic disorders, but the underlying mechanisms have not been fully clarified. In the current work, pea (Pisum sativum L.) seed meal proteins (albumins, legumins and vicilins) were isolated, submitted to a simulated gastrointestinal digestion, and the effects of their hydrolysates (pea albumins hydrolysates (PAH), pea legumins hydrolysates (PLH) and pea vicilin hydrolysates (PVH), respectively) on 3T3-L1 murine pre-adipocytes were investigated. The pea vicilin hydrolysate (PVH), but not native pea vicilins, increased lipid accumulation during adipocyte differentiation. PVH also increased the mRNA expression levels of the adipocyte fatty acid-binding protein (aP2) and decreased that of pre-adipocyte factor-1 (Pref-1) (a pre-adipocyte marker gene), suggesting that PVH promotes adipocyte differentiation. Moreover, PVH induced adiponectin and insulin-responsive glucose transporter 4 (GLUT4) and stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, were up-regulated in 3T3-L1 cells treated with PVH during adipocyte differentiation. Finally, PVH exhibited PPARγ ligand activity. Lactalbumin or other pea hydrolysates (PAH, PLH) did not exhibit such effects. These findings show that PVH stimulates adipocyte differentiation via, at least in part, the up-regulation of PPARγ expression levels and ligand activity. These effects of PVH might be relevant in the context of the beneficial health effects of legume consumption in obesity-associated metabolic disorders.

Evaluation of Immunoreactivity of Pea (Pisum sativum) Albumins in BALB/c and C57BL/6 Mice.

Green pea (Pisum sativum) is a component of European cuisine; however, an estimated 0.8% of Europeans suffer from allergies to pea proteins. We examined the immunoreactive potential of pea albumins (PA) in BALB/c and C57BL/6 mice. Mice were orally gavaged with PA or glycated pea albumins (G-PA) for 10 consecutive days, in combination with an adjuvant. Both PA and G-PA increased PA-specific serum antibody titers to about 212 for anti-PA IgG, ∼27 for anti-PA IgA, and ∼27.8 for anti-PA IgA in fecal extracts (p < 0.001). On day 42 postexposure, the antibodies titers decreased and were greater in BALB/c compared to C57BL/6 mice (p < 0.05). Distribution of CD4+ and CD8+ T cells in lymphoid tissues presented strain-specific differences. PA was found to induce lymphocyte proliferation; however, G-PA did not. Both PA and G-PA changed CD4+ and CD8+ T cells percentages in some lymphoid tissues; however, this did not impact cytokines production by splenocyte cultures evidenced by the stimulation of Th1, Th2, and Th17 cells. The observed immunomodulatory properties of PA and G-PA and lack of a sign of allergic reaction render them suitable for supplements in personalized diets, but further research is needed to precisely understand this activity.

Gelation behaviors of denaturated pea albumin and globulin fractions during transglutaminase treatment

The behavior of pea albumins (Alb) and globulins (Glob) in a denatured state toward microbial transglutaminase (MTGase) treatment was studied by SDS-PAGE analysis, free amine group determination, dynamic rheology and confocal laser scanning microscopy. The denaturation of pea proteins by chemical reduction with dithiothreitol (DTT) or by thermal treatment at 80 °C enhanced the enzymatic crosslinking degree of both protein isolates with greater crosslinking for the Glob fraction. The chemical denaturation affected preferentially the participation in crosslinking reaction of legumin acid subunits (40 kDa) for Glob sample and albumin polypeptides PA2 (26 kDa) for Alb sample, whereas the heat treatment led to complete polymerization of 55, 35 and 30 kDa vicilin polypeptides for pea globulins as shown by SDS-PAGE analysis. Up to 10 wt% concentration, the Alb fraction was not able to form MTGase crosslinked gels whatever the initial native or denaturated state. Compared to the native state, chemical and thermal denaturation of Glob fraction before enzymatic treatment led to the formation of weaker and stronger viscoelastic gels respectively. These contradictory results indicated that the enzymatic crosslinking reaction is highly related to polypeptides composition and conformation of proteins and the use of denaturation as a strategy to enhance gel forming properties by transglutaminase treatment has to be used with caution in the case of plant proteins.

Probiotic fermented milks made of cow's milk, goat's milk and their mixture

Probiotic fermented milks made of cow's milk, goat's milk and their mixture

Immune response in Balb/C vs C57BL/6 mice during oral immunization with pea albumins (Pisum sativum)

Immune response in Balb/C vs C57BL/6 mice during oral immunization with pea albumins (Pisum sativum)

Experimental immunology The effect of pea albumins on immune response in mice

Experimental immunology The effect of pea albumins on immune response in mice

Relationship between proportion and composition of albumins, and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.)

Peas provide an excellent plant protein resource for human diets, but their proteins are less readily digestible than animal proteins. To identify the relationship between composition and in vitro digestibility of pea protein, eight pea varieties with a wide range of protein content (157.3-272.7 g kg(-1)) were determined for the proportion of albumins and globulins, their compositions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in vitro protein digestibility (IVPD) before and after heat treatment using a multi-enzyme (trypsin, chymotrypsin and peptidase) method. The proportion of albumins based on total seed protein content decreased from 229 to 147 g kg(-1) as seed protein content increased from 157.3 to 272.7 g kg(-1), while the proportion of globulins increased from 483 to 590 g kg(-1). The IVPDs of eight raw pea seeds were 79.9-83.5%, with significant varietal variations, and those were improved to 85.9-86.8% by cooking. Albumins, including (pea albumins 2) PA2, trypsin inhibitor, lectin and lipoxygenase, were identified as proteolytic resistant proteins. Globulins were mostly digested by protease treatment after heating. The quantitative ratio of albumins and globulins, and the quantitative variations of albumin protein components, including lipoxygenase, PA2, lectins and trypsin inhibitors, appear to influence the protein digestibility of both raw and cooked pea seeds.

Effect of non-enzymatic glycosylation of pea albumins on their immunoreactive properties

The aim of this study was to determine the effect of non-enzymatic glycosylation of pea proteins on their immunoreactive properties. Extracted total pea albumins were glycated. No changes were found in molecular weight distribution of total pea albumins before and after glycation using size exclusion chromatography and SDS–PAGE methods. SDS–PAGE GLYCO test stained the glycated proteins and OPA method showed 15% progress in glycation. Glycated and unglycated pea albumins were orally and intraperitoneally administered to Balb/C mice. Serum specific IgG and IgA and sIgA were determined. No difference in serum specific IgG level was found after oral mice immunization with TA and GTA. In the presence of antigen SPL lymphocytes culture showed higher proliferation activity as compared to the culture without the antigen addition. The glycation does not change the immunoreactivity of proteins significantly. During the presented route of immunization with TA and GTA specific tolerance mechanism could be induced.

Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin and pea albumin 2.

A study was conducted to investigate the biochemistry of digestion of field pea (Pisum sativum L.) albumins and globulins in the stomach and along the small intestine of weaned piglets with a particular emphasis on the respective roles of these compartments in pea protein digestion. Twenty-four piglets were weaned at 28 d of age. They were allocated to 2 diets (control and pea) and 3 slaughter times (3, 6, or 9 h after the last meal) in a 2 x3 factorial arrangement of treatments in a randomized complete block design. Pea flour provided 30% of total dietary protein in the pea diet. The diets were fed for 2 wk after weaning. After slaughter, gastrointestinal tract (GIT) compartments were weighed, digesta were collected, and pH was measured. Digesta from the stomach and cranial, middle, and caudal small intestine (SI) were extracted for soluble proteins and analyzed for specific pea proteins using SDS-PAGE, immunoblotting, and mass spectrometry. Tissue weight of the whole GIT (P = 0.015), cecum (P <0.001), and colon (P <0.001) was greater in the pea diet. Digesta pH in the stomach and caudal SI was lower (P = 0.02) in the pea diet than the control diet. In the stomach, vicilin, lectin, and pea albumin 2 were not digested, whereas legumin was only partly digested. Legumin and vicilin were totally digested in the SI in less than 3 h. A resistant peptide of 15 kDa located at the N-terminus of pea albumin 2 was transiently detected at 3 h. A protein band at 20 kDa was consistently identified as lectin. It was present in high intensity in intestinal digesta of pea-fed piglets at all times after the meal compared with those fed the control diet (P <0.001). Various proteins of, presumably, endogenous origin displayed differential digestion patterns between the control and the pea-fed piglets (P<0.05). In conclusion, differences in digestion between specific pea proteins were observed along the GIT of piglets. They could be partly explained by differences in protein digestion in the stomach.

Influence of non‐enzymatic glycosylation (glycation) of pea (Pisum sativum) albumins on their enzymatic hydrolysis

AbstractThe aim of this study was to investigate the influence of non‐enzymatic glycosylation (glycation) on the susceptibility of pea albumins to pepsin hydrolysis. It was proved that the aqueous albumin extract is de facto a nucleo‐glyco‐metalloprotein complex. The non‐enzymatic glycosylation of pea albumins decreased the content of nucleic acids, hexoses and zinc, which bound to this protein. Glycated pea albumins were more susceptible to hydrolysis by pepsin than non‐glycated ones. Copyright © 2005 Society of Chemical Industry

Protein Interactions of Cheese Whey-Pea Flour Blends

Fresh cheese whey and whey ultrafiltrate were compared to water as an extractant for pea flour nitrogen. Whey was 18% less efficient than water at pH 8.0 and 48% less efficient at pH 6.8. Whey ultrafiltrate was only slightly more efficient than whey.Affinity columns were prepared by coupling isolated whey proteins, pea albumins or pea globulins to Sepharose 4B, and solutions of the same proteins were chromatographed on the various columns. At low ionic strength pea globulins showed considerable affinity for the whey protein column and also for the pea albumin column. Pea albumins showed little affinity for whey proteins. On chromatography of a pea protein solution containing both albumins and globulins on the whey protein column, it appeared that albumins were masking the binding sites of the globulins. Proteins retained by the affinity columns were readily eluted by increasing the ionic strength. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the chromatographic peak fractions indicated some tendency for selective adsorption of certain pea globulin subunits by the whey protein matrix.