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Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β‑Lactoglobulin Mohamed-Lazhar Chihi,†,§ Jean-luc Mession,† Nicolas Sok,† and Rémi Saurel*,† †

UMR Procédés Alimentaires et Microbiologiques (PAM), AgrosupDijon, Université Bourgogne Franche-Comté, 1 Esplanade Erasme, 21000 Dijon, France § Département de Technologie Alimentaire, Unité de Recherche et Développement de l’intendance, 16000 Alger, Algeria ABSTRACT: The present work investigates the formation of protein aggregates (85 °C, 60 min incubation) upon heat treatment of β-lactoglobulin (βlg)−pea globulins (Glob) mixtures at pH 7.2 and 5 mM NaCl from laboratory-prepared protein isolates. Various βlg/Glob weight ratios were applied, for a total protein concentration of 2 wt % in admixture. Different analytical methods were used to determine the aggregation behavior of “mixed” aggregates, that is, surface hydrophobicity and also sulfhydryl content, protein interactions by means of SDS-PAGE electrophoresis, and molecule size distribution by DLS and gel filtration. The production of “mixed” thermal aggregates would involve both the formation of new disulfide bonds and noncovalent interactions between the denatured βlg and Glob subunits. The majority of “mixed” soluble aggregates displayed higher molecular weight and smaller diameter than those for Glob heated in isolation. The development of pea−whey protein “mixed” aggregates may help to design new ingredients for the control of innovative food textures. KEYWORDS: pea globulin, β-lactoglobulin, heat denaturation, mixed aggregates, aggregate size, protein

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INTRODUCTION Owing to excellent yields, availability, and low-price production, the development of alternative plant protein sources in Europe such as pea seeds (Pisum sativum L.) may account for further prospects concerning a sustainable human food supply.1,2 In some respects, the increasing potential of legume proteins arose from the opportunity to replace ingredients from animal production, due to their functional properties in textured foodstuffs and also their nutritional quality and reduced allergenicity.3 However, one recent issue is to associate different sources of protein such as plant proteins and other proteins so as to formulate new ingredients or food products.4 Therefore, a better understanding of the interactions between plant proteins such as pea proteins and milk proteins and their assemblies is expected. In this paper, we propose the formation of mixed thermal aggregates between pea proteins and whey proteins for similar food applications to those already proposed for whey protein aggregates (gelation, stabilization of foam and emulsion, etc.).5 Proteind account for 20−30 wt % of pea seeds and are composed mainly of 7S/11S globulin (Glob, 50−60% of total) and albumin 2S (15−25%) protein classes.6 Pea globulins (Glob) are constituted of two main protein species, namely, legumin 11S and vicilin/convicilin 7S.1,6,7 Legumin 11S is hexameric homo-oligomer, of molecular weight (Mw) ∼ 360− 400 kDa. Each legumin subunit (∼60 kDa) is made up of disulfide-bonded acidic (∼40 kDa) and basic polypeptides (∼20 kDa). 8 Legumin subunit has approximately four methionine and between two and seven cysteine amino acid residues.9,10 Vicilin 7S is trimeric, of average Mw ∼ 150 kDa ; the main vicilin subunit (∼50 kDa) can undergo in vivo proteolysis according to two potential cleavage sites, resulting in smaller vicilin fragments. The vicilin polypeptides are depleted of sulfur amino acids.7,9−11 Convicilin 7S is a third © XXXX American Chemical Society

storage protein (∼290 kDa), consisting of subunits (∼71 kDa) associated into trimers or tetramers.10,11 Whereas whey proteins (WPs) were previously considered as a byproduct of cheese manufacture, these are well-spread in textured foodstuffs owing to excellent thickening and gelling properties. Whey protein isolates (WPI) contain mainly βlactoglobulin (βlg) (44−70%),12 existing as a dimer at room temperature and at pH in the range of 3−7.5, of which monomers have a Mw of ∼18 kDa. βlg has two disulfide bridges and one free thiol group.5,13,14 WPs display well-balanced essential amino acid composition, among them sulfurcontaining ones.13 Thus, it appeared that the combination of WPs with legume proteins may help to decrease the sulfur deficiency of the latter ones.15 In this regard, mixing both protein sources could match the nutritional, functional, Western consumer acceptability, and sustainable requirements expected for innovative food products. For instance, limitation may result from the interactions between dissimilar proteins as induced by usual food processing, such as heat treatment. Heat-induced aggregation is one of the most important properties of food proteins. Heat-set gelation of globular proteins has been reported extensively1,2,16 to produce food products displaying various structural and textural features.5,17 Thermal gelation of pea proteins was documented recently. According to the authors, pea protein gelation was affected by several factors, among others, cultivar, extraction procedure, solvent parameters, and heating procedure.18,19 By thermal denaturation, globular proteins at level below the minimal Received: January 7, 2016 Revised: March 16, 2016 Accepted: March 20, 2016

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DOI: 10.1021/acs.jafc.6b00087 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

induced “mixed” aggregates from laboratory-extracted Glob and βlg fractions was not previously investigated. Therefore, the heated protein mixtures were extensively characterized in terms of molecular interactions involved, particle size, and protein aggregate composition as related to different βlg/Glob weight ratios applied in the mixtures.

gelling concentration could yield soluble aggregates in predetermined solvent conditions, that is, at neutral pH and low ionic strength applied. Numerous interactions are involved in the denaturation/aggregation process, such as covalent and noncovalent interactions.5,13,16 A high level of electrostatic repulsions in the absence of salt and at pH higher than the isoelectric pH is required to prevent rapid protein aggregation via noncovalent interaction subsequent to denaturation. Upon denaturation, the protein unfolded (step 1) and exposed buried reactive groups (step 2), previously in the core of the globular structure. Within a third step, the unfolded proteins associated more or less linearly, depending on the balance between electrostatic repulsions and exposed hydrophobic groups.5,13,16 In particular, βlg thermal aggregation generally occurs in three stages: first unfolding and dissociation of the βlg dimmers into reactive monomers, then exposure of free thiolate (sulfhydryl) groups, resulting in the reassociation of the monomers into intermediate aggregates via intermolecular sulfhydryl−disulfide bond exchange reactions. Thereafter, these primary aggregates reacted through covalent and noncovalent (mainly hydrophobic) interactions, to ultimately form high-Mw and soluble, spherical aggregates with a compact apparent diameter at pH 200 kDa) under N or NR conditions were estimated by calculating the total lane intensity ratio (1 − IN/INR or 1 − INR/IR, respectively) for each sample under NR and R conditions on separate lanes of the same electrophoresis gel. Under R conditions, it was considered that the whole polypeptide could migrate; hence, total intensity (sum of individual band intensities) was attributed to total protein amount deposited. Each sample was analyzed in duplicate. Dynamic Light Scattering (DLS). A PSS-380 Nicomp submicrometer particle size distribution analyzer (Santa Barbara, CA, USA) was used to estimate molecule size of unheated/heated protein samples in diluted regime. The DLS instrument consisted of a beam laser (632 nm wavelength) at a fixed scattering angle of 90° and a photodetector connected to a correlator CW388. Protein samples were diluted adequately at 0.1 wt % with deionized water, filtered through 0.22 μm cellulose membrane (Millipore Corp.), and poured in a 1 cm3 glass vessel. At least five measurements of 20 min data acquisition time for each protein sample were performed at 25 °C. Particle size distribution by means of apparent hydrodynamic diameter (Dh) was obtained by the Nicomp Analysis as supplied with the software (CW388 Application, v. 1.68); as the size of protein aggregates in solution could be polydisperse, the apparent diameters of samples will be given more preferably as multimodal distribution (Nicomp 380 DLS User Manual, 2006). The distribution of Dh by intensity was represented. The data were interpreted qualitatively because large particles (>60 nm) scattering light more strongly than smaller ones would increase intensity at a given angle of detection. Moreover, the sensitivity toward fluctuations in shape, dispersity, and internal dynamics of large aggregates would influence the estimated apparent size of particles. Molecular Weight Determination. The Mw distribution of protein samples was evaluated using a Shimadzu high-pressure liquid chromatography system (Shimadzu Corp., Kyoto, Japan) equipped with an isocratic pump (Shimadzu LC-20AT) and UV−visible detector (Shimadzu SPD-20AV) mounted with a TSK gel G6000 PWxl size exclusion column (7.8 mm internal diameter × 30 cm length, Tosoh Bioscience, Stuttgart, Germany). Samples were loaded manually. The column was pre-equilibrated at 25 °C with mobile phase, consisting of 50 mM phosphate buffer and 0.05 M NaCl, pH 7.2, previously filtered through 0.22 μm filters and degassed. The flow rate was 0.4 mL min−1. The column was calibrated with a wide Mw protein standard from Sigma-Aldrich (MWGF1000-1KT) and GE Healthcare. The estimated Mw separation range of the column was between 10 and 8 × 103 kDa, based on the supplier’s prescription. The total column volume was 14.34 mL, which void volume (V0) was ∼4.3 mL. Protein samples were filtered through a 0.45 μm membrane (Millipore Corp.) and injected manually into the system with a syringe-loading sample loop injector (100 μL). Absorbance was recorded at 280 nm, and peaks were analyzed with the LC Solution software (v. 1.25, Labsolutions, Shimadzu). All samples were measured in triplicate. Additionally,

stock solutions was adjusted to 7.2 with 0.1 M NaOH. Protein isolates were stirred at 4 °C and left 24 h for complete hydration. After centrifugation (12000g, 20 min, 25 °C), supernatants for each stock solution were pooled and filtered using a 0.45 μm cellulose membrane (Millipore). It was checked that the amount of protein loss was negligible during this procedure (data not shown). Different mixtures at 2 wt % total protein concentration were prepared by mixing together the protein stock solutions for 2 h at 25 °C and by applying several βlg/Glob weight ratios (0/100, 30/70, 50/50,70/30, 100/0). For each mixture composition, samples were poured in hermetically sealed glass tubes, placed in a temperature-controlled water bath previously equilibrated at 40 °C, then heated at 1 °C/min from 40 to 85 °C, incubated at 85 °C for 60 min, and rapidly cooled in ice for 10 min, according to our previous work on pea globulin aggregation.33 If it occurred, insoluble material was removed by centrifugation (12000g, 20 min, 25 °C). Prior to each analysis, the heated protein samples were tempered at 25 °C. The DSC analysis of the heated mixtures displayed no endothermic peak, indicative of complete protein denaturation. The percentage of soluble protein aggregates was determined after centrifugation (12000g, 20 min, 25 °C) as the ratio of water-soluble nitrogen to total nitrogen, determined by Kjeldahl method; soluble protein in heated mixtures accounted for ∼98% of total protein, regardless of the βlg/Glob ratio. Single protein samples and their mixtures were further characterized as listed below. Surface Hydrophobicity. Protein surface hydrophobicity was determined with the fluorescence probe 1-anilinonaphthalene-8sulfonic acid (ANS; Sigma Co., St. Louis, MO, USA) according to the modified method of Kato and Nakai34 by Rayan12 and Karaca.35 Soluble protein samples were diluted in a range of concentrations of 0.004−0.02 wt % in a 10 mM Na2HPO4 buffer, pH 7.2. Twenty microliters of the 8 mM ANS stock solution as prepared in the same buffer was added to 4 mL of each protein sample. Samples were poured into quartz cuvettes, after each sample had been kept in the dark for 15 min; the fluorescence intensity was measured using a FluoroMax-4 spectrophotometer (Horiba Jobin Yvon Inc., Edison, NJ, USA) with excitation and emission wavelengths at 390 and 470 nm, respectively. Blanks were protein samples without ANS and buffer with ANS. The surface hydrophobicity index (H0) was calculated by linear regression analysis of the plot of fluoresence intensity against protein concentration. Assays were performed three times. Sulfhydryl Content Determination. The free sulfhydryl (S−) and disulfide bond (S−S) contents of the protein samples were measured using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), namely, Ellman’s reagent.36 For S−free content determination, protein solutions at 2 wt % concentration were extensively dialyzed against a 0.1 M phosphate buffer at 4 °C, pH 7.5 (ratio protein solution-to-buffer 1:20). The reaction mixture in the phosphate buffer was prepared by mixing 650 μL of the protein sample with 750 μL of guanidium hydrochloride (3 M GdnCl) and 100 μL of DTNB solutions (1 mM), stirred vigorously, and kept in the dark for 10 min at 25 °C. Absorbance was measured at 412 nm using a molar extinction coefficient ε for DTNB of 12900 M−1/cm, as calculated from calibration curves with N-acetylcysteine (NAC) in the range of 0−60 μM. For S−S estimation, total sulfhydryl groups S−total (S−free + S−S) of the different protein samples were recovered by treatment with 20 mM DTT added as powder, for 2 h at 25 °C. Further extensive dialysis with degassed phosphate buffer allowed the elimination of the excess of DTT, with limited risk of reoxidation of the released S−. The S−S content was calculated as follows (eq 1):

[S−S] = −

([S−]total − [S−]free ) 2

(1) −

The free S and S−S contents were expressed as μmol S /gprotein and μmol S−S/gprotein, respectively. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE). Polypeptide compositions of the different protein samples were analyzed by tris−glycine SDS-PAGE on slab gels (4% stacking and 12% (bis) acrylamide (w/v) separating gels, pH 6.8 and 8.9, respectively), as described by Laemmli.37 Protein samples (2 wt %) were diluted at a concentration of ∼5 mg/mL with the sample C


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Journal of Agricultural and Food Chemistry aggregate peaks were collected, dialyzed against water, and freezedried. The aggregate composition was further analyzed by SDS-PAGE. Statistical Analysis. Values given in the tables and figures are the means of triplicates. Significance of differences among samples was evaluated by one-way ANOVA test set at the 5% p level, using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA).

extraction process may have brought hydrophobic groups, buried in the heart of the proteins, to the surface35 and resulted in partial dissociation of native polypeptidic legumin subunits.40 An increase in H0 could result from hydrophobic groups exposure to the surface of thermally unfolded proteins. For single-Glob and single-βlg samples heated in isolation, H0 increased by about ∼1.5- and ∼3.5-fold, as compared to unheated samples, respectively. With regard to βlg, this was in agreement with Zhu et al.41 For the same heating procedure and solvent conditions, the surface hydrophobicity of heated single βlg was ∼1.2 times higher than that for pea Glob. The difference between proteins may be due to dissimilar amino acid composition as well as a heat-induced change in protein structure upon denaturation and aggregation.13,40−42 In an earlier study, pea protein denaturation resulted in the breakup of the oligomeric structure, and the released subunits were shown to reassociate via exposed reactive groups, predominantly by nonspecific hydrophobic interactions.9,22,23 In some cases, molecular rearrangements during protein aggregation were hypothesized to hinder some hydrophobic areas and consequently decrease the H0 values of pea Glob after heat treatment. Regardless the βlg/Glob ratio, the soluble thermal aggregates in the mixtures displayed higher H0 values than the unheated single-protein solutions. The H0 values increased significantly but rather weakly with the βlg/Glob ratios. The significant increase in H0 values after heat treatment could indicate the intensive participation of hydrophobic interactions in the formation of thermal aggregates from the mixed proteins, as the thermal aggregation mechanism of globular protein is recognized to be controlled mainly by hydrophobic interactions.12,34 Quantification of Disulfide Bridges. To highlight the implication of disulfide bridges in the formation of aggregates made up with Glob, βlg, and/or mixtures of the two, free sulfhydryl groups and disulfides bridges were quantified using the Ellman method.36 Table 1 displays the free sulfhydryl group and S−S group concentrations in native Glob and βlg solutions and in heated single-protein solutions and their mixtures. With regard to βlg solutions, protein aggregation led to a decrease and an increase in free S− (from 42 to 17 μmol S−/gprotein) and S−S (from 102 to 114 μmol S−S/gprotein), respectively; this result is in agreement with the work of Funtenberger et al.43 The sulfhydryl groups of whey proteins are buried in the tertiary structure of the native molecule. Heat denaturation renders these groups accessible, resulting in thiol oxidation and S−/S−S exchange reactions and thus formation of disulfide bridges between protein molecules in the aggregates.13,14,44,45 The free S− group content (approximately 17 μmol S−/gprotein) of single-βlg aggregates was lower than that obtained by Liu et al.45 for βlg thermal aggregates at pH 7, that is, ∼28 μmol S−/ gprotein of accessible thiol after heating at 85 °C for 15 min. This may suggest that the concentration and the prolonged heat denaturation at 85 °C for 1 h in the present study were more favorable to S−/S−S exchange reactions. With regard to Glob, legumin is the main source of sulfurcontaining amino acids,18 whereas convicilin may contain one or two cysteine residues per molecule and vicilin is devoid of it.7,8 The free S− group contents measured for native and Glob thermally treated samples were lower and comparable with that reported by Mession et al.33 Thermal aggregation of globulins led to a >2-fold decrease in S−S content, whereas the S− content increased markedly. As observed previously by authors above for legumin-enriched fraction, the increase in S− free

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RESULTS AND DISCUSSION Protein Composition of Isolated Fractions. The purified βlg isolate prepared from the WPI was composed of ∼92 wt % protein, ∼3 wt % ashes, and ∼0.8 wt % fats on a dry basis. The protein composition of the βlg extract was verified using SDSPAGE (data not shown). Patterns exhibited mainly a band of monomeric βlg and one other band corresponding to negligible traces of non-native dimeric βlg, which confirmed that the purification was performed correctly. With regard to the Glob isolate, protein and fat contents were around 94 and 0.05). nd, not determined. bAs described under Determination of Surface Hydrophobicity cAs described under Quantification of Disulfide Bridges.

hydrophobic moieties of the soluble protein molecules, displaying more affinity for aromatic amino acids.38 Extracted Glob had much higher H0 values than βlg, as the former one exposed more hydrophobic residues. Surface hydrophobicity is related to protein structure that could depend on solvent parameters (pH, ionic strength, temperature) and extraction procedure. According to the literature, the legumin Lβ polypeptide is more hydrophobic than Lα and located in the core of the legumin hexameric structure.39,40 Alkaline extraction followed by acid precipitation during the Glob preparation and D


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sample under R and NR conditions (lanes 13 and 12); ∼10% of initial legumin subunits would be involved in aggregation via new disulfide bridges. This result is consistent with the previous S−/S−S content determination, which showed the nonparticipation of many released free S− groups upon heat denaturation in aggregate formation. As ∼90% of legumin subunits migrated in lane 12, these polypetides form aggregates through noncovalent bonds. Mori et al.22 reported similar results regarding the formation of 11S soy aggregates, mainly via hydrophobic interactions, at a protein concentration of 0.5%. However, when a higher soy protein concentration of 5% was used, the formation of 11S soluble aggregates was also found to be governed by the reassociation of acidic and basic subunits through covalent bonds. The densitometric analysis of lanes 12 and 13 showed that the migration patterns of 7S polypeptides were identical under both conditions. On the basis of these results, we can confirm that the interactions generated during denaturation and aggregation, if occurred, of vicilin subunits are of noncovalent nature.9,23 By calculating the relative intensity between NR and R conditions from densitometric analysis of the single βlg samples, it was observed that around 70% of total protein content may be associated into high Mw covalent aggregates via new disulfide bonds, in agreement with previous works.14,20,44 Analysis of the electrophoretic patterns of protein mixtures at weight ratios βlg/Glob of 30/70, 50/50, and 70/30 first shows the presence of an L(αβ) band (60 kDa) under N condition, representing only ∼5% of the total legumin in the 30/70 mixture. This minor legumin band is no longer visible in lane 2 with SDS. This L(αβ) subunit is therefore maintained by a noncovalent bond and would coexist with the soluble aggregates formed, as suggested by Mession et al.23 It has also been shown that after heat-induced denaturation of bean legumin, the subunits L(αβ) recombine, forming aggregates through noncovalent bonds, with no peculiar involvement of sulfhydryl groups.24 For the other mixtures in N conditions (lanes 4 and 7, respectively), this minor legumin fraction is not observed, which could mean that this fraction formed high Mw aggregates with other legumin and/or βlg fractions when mixed together. The relative intensity between NR and R conditions corresponded to ∼32, 15, and 6% for Lα bands and 15, 13, and 4% for Lβ1, 2 bands at the βlg/Glob weight ratios of 30/70, 50/ 50, and 70/30, respectively. By comparison to single-Glob solution, where disulfide-bonded legumin accounted for not more than 10%, more legumin subunits were involved in the formation of thermal aggregates by covalent interactions (∼70−95% depending on the βlg/Glob weight ratio). Moreover, these legumin fractions participated more intensively to the formation of disulfide-bonded aggregates (Mw > 200 kDa) when the βlg/Glob increased. In addition, the nonmigrating disulfide-bonded aggregates may account for ∼33, 37, and 55% of total polypeptides in the 30/70, 50/50, and 70/30 mixtures, respectively. All of the vicilin subunits were observed in lane 1 (N condition), but with a lower intensity of ∼35% in relation to the same polypeptides in lane 2. Possibly these vicilin polypeptides did not participate in the formation of protein aggregates at low protein concentrations and at low ionic strength. Similar behavior was reported by Li et al.17 for soy 7S protein in heat-treated single-globulin solution at low concentration (2% w/w) and low ionic strength (0 mM). The same results were obtained with 50/50 and 70/30 mixtures (lanes 4, 7), with a decrease in intensity of the vicilin fractions from ∼22 to 13% in lanes 6 and 9, respectively, which is

content would originate from the simultaneous disruption of preexisting disulfide bridges and random aggregation of unfolded polypeptide chains by nonspecific interactions. As the accessibility of S− free groups would be decreased in the amorphous aggregates, a few released S− groups would be involved in the formation of new disulfide bonds. With regard to heated mixtures, increasing the βlg/Glob weight ratio led to an increase and a decrease in S−S and S− contents, respectively. These variations seemed almost proportional to the presence of βlg in the mixture. However, it was not possible to deduce if disulfide bonds were formed between the two protein sources during the formation of these heat-induced mixed aggregates, as the Ellman method is a global method. SDS-PAGE and Densitometric Analysis. SDS-PAGE was used to characterize heat-induced change in protein composition within samples. Figure 1 shows the polypeptide

Figure 1. SDS-PAGE pattern under “native” (N) condition, NR and R conditions of the heated single-βlg, single-Glob samples and the βlg/ Glob mixtures prepared at different weight protein ratios, in 5 mM NaCl and pH 7.2. Lanes: 1, 2, and 3, ratio 30/70 under N, NR, and R; 4, 5, and 6, ratio 50/50 under N, NR, and R; 7, 8, and 9, ratio 70/30 in N, NR, and R; 9 and 10, heated single-βlg under NR and R; 11 and 12, heated single-Glob sample under NR and R; M, molecular mass markers (kDa); L(αβ), legumin; Lα, legumin acidic polypeptide; Lβ, legumin basic polypeptide; V, vicilins; CV, convicilin; LP, lipoxygenase; βlg, β-lactoglobulin; Glob, globulin.

composition of the soluble thermal aggregates from βlg, Glob, and mixtures of the two at the same initial protein concentration for all samples. For heated samples, the presence of high-Mw aggregates (>200 kDa) in N conditions (lanes 1, 4, 7) and NR conditions (lanes 2, 5, 8, 10, 12) was observed; the disappearance of their related bands in R conditions was indicative of new, heat-induced disulfide bonds, possibly involving Lα, Lβ, and βlg within self-aggregates or possibly mixed ones (lanes 3, 6, 9, 11, 13). The electrophoretic pattern of heated Glob sample revealed deposits of high molecular weight aggregates (>200 kDa) in lane 12 in the stacking gel. The Lα, Lβ1, and Lβ2 subunits were partially affected by aggregation through covalent bonds. Indeed, the intensity of corresponding bands increased slightly by ∼11, 10, and 11% for subunits Lα, Lβ1, and Lβ2, respectively, by comparing the same E


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Figure 2. Particle size distribution measured by DLS at 25 °C for (a) unheated single-protein (βlg, Glob) samples and (b) heated (85 °C for 60 min) single-protein samples and their mixtures at different βlg/Glob protein weight ratios, at 2 wt % total protein concentration and in 5 mM NaCl, pH 7.2, then diluted to 0.1% in deonized water.

Table 2. Particle Size Distribution in Terms of Hydrodynamic Diameter Dh for (a) Unheated and (b) Heated Single-Protein Samples and βlg/Glob Mixturesa particle diameter (Dh) (nm) size distribution

Glob

30/70

50/50

βlg

70/30

(a) Unheated particle 1 % particle 2 %

16.9 56 94.5 43.9

± ± ± ±

5.6 1.5 1.5 0.4

particle 1 % particle 2 %

69.3 63.1 151.9 36.9

± ± ± ±

5.6 a 1.5 9.8 a 0.4

nd nd nd nd

nd nd nd nd

6 ± 0.4 100 * *

nd nd nd nd

(b) Heated 43.2 28.9 111 71.1

± ± ± ±

0.2 b 2.4 10.1 b 2.2

37 15.6 95 84.4

± ± ± ±

3.6 bc 1.2 8.32 c 0.8

27 2.3 94.9 97.7

± ± ± ±

0.3 c 0.2 0.1 c 0.6

* * 38 ± 0.07 d 100

“Particle 1” and “Particle 2” ranges were attributed to small size particles and large size particles. Percentage of the particles (%) by means. All results are given as the mean ± standard deviation, calculated from at least five repetitions. Means bearing the same letter (ad) are not significantly different (p > 0.05). nd, not determined. An asterisk indicates the absence of a peak. a

on the βlg/Glob ratio. Contrary to other 7S polypeptides, the

proportional to the decrease in the Glob content in the mixtures. Convicilin subunits had a peculiar and previously undescribed behavior because an increase of the band intensity under R conditions was measured, indicating they participated at 20−30% in aggregation through covalent bonds depending

convicilin subunit contains a cysteine residue, which could be involved in a disulfide bond with other proteins. As this was not observed for single-Glob solution, it could be hypothesized that F


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Figure 3. SEC-HPLC profiles of proteins prepared for (a) unheated single-protein (βlg, Glob) samples and (b) heated (85 °C for 60 min) singleprotein samples and their mixtures at different βlg/Glob protein weight ratios, at 2 wt % total protein concentration and in 5 mM NaCl, pH 7.2. Spectra were divided into several elution ranges, according to Mw distribution (refer to Table 3 for fraction assignment). β, unheated βlg; G1−5, unheated Glob sample; β′1, heated βlg; G′1−4, heated Glob; βG′1−3, heated βlg/Glob mixtures. (c) Calibration curve of the column with a wide range of Mw standards: blue dextran (2000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa). The flow rate was 0.4 mL min−1.

convicilin interacts preferentially with βlg through covalent interaction. Moreover, the level of covalent aggregation of βlg in admixture with Glob was not found to differ with the heated single-βlg sample (∼70% of initial βlg amount). According to these results, greater involvement of legumin subunits in the formation of aggregates by covalent bonds was evidenced in the case of mixtures with respect to single-Glob samples, whereas the level of covalent aggregation of βlg seemed unaffected. The implication of βlg in the formation of mixed aggregates may be hypothesized even if the results do not exclude the simultaneous formation of pure aggregates of each protein. These results are in agreement with the previous data based on the S− and SS determinations, confirming the implication of covalent bonds between βlg subunits and/or legumin subunits upon heating of the mixtures. Determination of Soluble Aggregate Size Using DLS. Particle size distribution by means of hydrodynamic diameter (Dh) of unheated/heated protein samples was measured using DLS. The data are summarized in Figure 2 and Table 2. The apparent Dh for native βlg was ∼6 nm with a monomodal distribution, in agreement with reported data.5,16 After heat

denaturation, the increasing contribution of large particles may be attributed to the βlg aggregates, of apparent size centered at around 40 nm. In this study, at pH 7.2 (>pHi of βlg), this Dh mean value for the aggregates formed can be attributed to (i) an increase in the intermolecular repulsive charge, which generally limits overaggregation and stops aggregates growing,44 and (ii) a prolonged heating time, leading to the formation of small nonsedimenting aggregates characterized by a Dh ≤ 50 nm.5,21,46 In the chosen experimental conditions, the results obtained using DLS may correspond to a intensive conversion of native proteins into one population size of aggregates.20 In contrast, size distribution of native Glob evidenced a bimodal distribution; particles around 15 nm may correspond to 7S/ 11S oligomers, whereas those at 100 nm could be aggregated protein particles formed during Glob preparation or initially present.17 After heating, the single-Glob sample displayed as well bimodal distribution, with two peaks centered at ∼70 and ∼150 nm; these were attributable to intermediate and large-size denatured pea protein aggregates, respectively. These results were similar to those measured elsewhere for soy protein aggregates by laser light scattering (LLS).17 However, Munialo G


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Table 3. Percentage of Integrated Area from SEC-HPLC Spectra at 280 nm, of Protein Fraction, As Presented in Figure 3a,ba (a) Native Proteins relative Mw (kDa) 660−443

443−43

43−29

29−13.7

G1

G2

G3

G4

G5

10.6 ± 2.9 35 ± 3.2

11.4 ± 2.10 17 ± 1.1

12.3 ± 1.8 14 ± 2.1

13.5 ± 1.3 10 ± 1.3

Glob

Evb %c

9.1 ± 3.4 23 ± 1.6

βlg

Ev %

−d −

600

600 ≫ Mw > 75

G′1 Glob

Ev %

βlg

Ev %

8.6 11.9 β′1 7.7 100

± 1.15 ± 1.4 a ± 1.2 ± 2.7 e

75−29

Mw > 2000

2000 ≫ Mw > 200

75−29

βG′1

βG′2

βG′3

7.4 47.5 7.5 66.5 7.6 78.2

± ± ± ± ± ±

1.8 1.9 b 1.5 2.3 c 2.7 0.9 d

9.7 32.3 9.7 20.1 9.7 13.3

± ± ± ± ± ±

2.5 2.3 b 1.2 1.4 c 0.8 2.5 d

10.9 20.2 10.8 13.4 10.82 8.5

± ± ± ± ± ±

1.6 0.5 b 0.50 0.9 c 2.3 1.4 d

a Spectra were divided into five protein fractions according to their elution volume range. The relative peak area (%) was defined as the peak area in each fraction, relative to the total integrated area of the spectrum (taken as 100%). All of the results are given as the mean ± standard deviation, calculated from at least three spectra. According to the sample, the fractions were (a) native proteins Glob (G1−5) and βlg (β) and (b) soluble aggregates Glob (G′1−4), βlg (β′1), and βlg/Glob mixtures (βG′1−3). Mean values bearing the same letter (a−e) are not significantly different (p > 0.05). An asterisk indicates the absence of a peak. bEv, elution volume (mL). cRelative percentage of protein fraction (peak area %). d−, not identified.

demonstrated before by SDS-PAGE or by S−/S−S content determination. Indeed, Schmitt et al.21 indicated that at neutral pH and in the absence of salt, the formation of disulfide bonds between βlg aggregates could limit and thus decreased the size of the aggregates. High-Performance Size Exclusion Chromatography (SEC-HPLC). The SEC-HPLC analysis was performed for the unheated/heated protein samples. Spectra are displayed in Figure 3a,b, and peak analysis is presented in Table 3. The elution profile of native βlg produced a single large peak corresponding to a Mw between ∼45 and 14 kDa in the calibrated column. This may be related to the presence of both dimeric and monomeric forms of βlg of calculated Mw of 36 and ∼18 kDa, respectively. After heating, there was an increasing contribution of high Mw compounds, related to the formation of large and soluble βlg aggregates (Mw ≫ 2000 kDa), comparable to that reported elsewhere.20,44,47 The native Glob elution profile exhibited two peaks eluting at volumes ∼9 and 10.5 mL, respectively, whereas the second one was shouldered toward lower elution range ( 600 kDa); G′2 represents Mw fractions between 75 and 600 kDa, which are considered as low- and medium-size aggregates; G′3 and G′4 can be attributed to heat-dissociated and nonaggregated subunits of ∼13.9−75 kDa. This result is in agreement with the SDS-PAGE results, which also highlighted that some heat-dissociated globulin subunits are not involved in aggregation. The results obtained are comparable to those reported by Mession et al.23 concerning Glob soluble aggregates. The authors indicated that the first population had a Mw of >700 kDa, which is consistent with our study for G′1 (Table 3). It seems that better separation and Mw distribution of the protein aggregates and fractions were obtained in relation to Mession et al.23 This could be due to the properties of the broader range separation column selected and the UV wavelength (280 vs 215 nm) retained, with which the response is more sensitive for aromatic I


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convicilin band, indicating that the latter is incorporated in the first two fractions (βG′1 and βG′2). Figure 4b shows the SDSPAGE pattern of the protein fractions corresponding to the peaks of βG′1, βG′2, and βG′3 aggregates for the βlg/Glob weight ratio 70/30. As previously for the 50/50 ratio, a deposit of aggregates in the stacking gel was observed in nonreducing conditions (lanes 8, 10). The addition of DTT (reducing conditions) revealed the Lα, Lβ, and βlg subunits (lane 7, 9); these bands were more intense for βG′1 than for βG′2. The band intensities of the βG′3 migration pattern were very low in relation to those obtained for the same fraction collected with a βlg/Glob weight ratio of 50/50. These results were in accordance with the SEC-HPLC relative signal intensity observed for the three peaks (Table 3). All of these results show the participation of the two types of proteins in the formation of high molecular weight βG′1 aggregates and medium-size βG′2 aggregates. The formation of these aggregates seems to be governed by covalent bonds involving disulfide bridge exchanges between βlg molecules and/or legumin/convicilin subunits and noncovalent interactions with other remaining globulin fractions (convicilin and vicilin subunits). Hypothetical Mechanism of the Thermal Aggregation of Both Proteins in Admixture. The aggregation of globular proteins depends on the equilibrium between their net surface charge and the attractive hydrophobic interactions following heat denaturation of these proteins.22 At pH 7.2, both proteins are negatively charged, thus repulsive. At neutral pH, the simultaneous heat denaturation of the two proteins in the mixtures resulted in the proteins unfolding by the loss of both quaternary and tertiary structures. At the same time the exposure of free thiol groups contributes to intermolecular disulfide bridge exchanges between βlg monomers and/or mainly legumin subunits (Lαβ) dissociated by the heat treatment.33 Finally, the new oligomers formed may interact, via noncovalent interactions (hydrophobic and/or electrostatic), incorporating the other nonaggregated Glob subunits (legumin, convicilin, vicilins), producing primarily high molecular weight soluble aggregates with a smaller hydrodynamic diameter (∼90−110 nm) than those formed by globulins alone (∼150 nm). Covalent interactions, in particular S−S bridge exchanges provided by βlg during the simultaneous heat denaturation of the two proteins, should play a role in the formation of these denser aggregates. An enhanced participation of some globulin polypeptides (legumin and also convicilin) through covalent bonds was also observed in the heated mixtures, giving substance to the formation of crossed covalent interactions between the two types of protein. The formation of mixed aggregates is assumed even if it was not possible to exclude the coexistence of pure aggregates of each protein with similar sizes. As the presence of βlg in the mixtures seemed to govern the particle size distribution and the molecular weight of the aggregates, this work proposes a new strategy for producing soluble aggregates containing plant proteins with controlled meso-structural properties. Future works should investigate the technofunctional properties of these mixed aggregates as well as their effects in terms of nutrition (susceptibility to proteolytic enzymes of the gastrointestinal tract, allergenicity, etc.). As short-term application, the formation of acid cold-set gel to design new food products incorporating pea protein will be presented in a forthcoming paper.

(βG′1), as well as a decrease in the proportion of intermediate aggregates (βG′2) and a decrease in the concentration of heat dissociated protein fractions not involved in aggregation (βG′3) (Figure 3b and Table 3). The decrease in βG′2 and βG′3 can be explained by the simultaneous participation of the latter two fractions in the formation of high molecular weight aggregates and/or intermediate aggregates (Table 3). According to these results, the molecular weight of the G′1 fraction aggregates decreased as the proportion of βlg in the mixtures increased: Mw (βG′1) 30/70 > Mw (βG′1) 50/50 > Mw (βG′1) 70/30 > Mw β′1 > Mw G′1. The comparison of these SEC-HPLC results with those obtained using DLS showed that a higher proportion of βlg in the mixtures resulted in (i) lower heterogeneity in the particle size distribution and a decrease in the average (Dh) of the aggregated particles and (ii) a decrease in the Mw of the aggregates formed. As described in the literature,18,19,22,23,33 the formation of soluble globulin aggregates is governed mainly by hydrophobic interactions as well as the formation to a low extent of new disulfide bridges for the legumin subunits, leading to the formation of random, disordered macroaggregates corresponding to a high Dh and a molecular weight >8 × 103 kDa. For βlg, the aggregates formed were particles of high molecular weight (8000 kDa > Mw ≫ 2000 kDa), but appeared to have a lower Dh and therefore a more compact structure. We can therefore suggest that the presence of low concentrations of βlg in the mixture (200 kDa) in the stacking gel (lanes 2, 4) and the migration of convicilin subunits, vicilins, and βlg, the latter being of low intensity. In reducing conditions (lanes 1, 3), all of the Glob subunits were observed with the dissociation of legumin into Lα and Lβ and the presence of very high intensity βlg. The convicilin band seemed also enhanced. These results indicated that mixed aggregates between subunits would be formed mainly by covalent interactions involving disulfide bond exchanges between βlg and/or the Lα and Lβ subunits and to a lesser extent convicilin subunits. A decrease in the intensity of the Lα, Lβ, and βlg bands in the βG′2 fraction was observed in relation to the βG′1 fraction, which could mean that many of these polypeptides were predominantly incorporated in the βG′1 fraction. The SDS-PAGE migration pattern of the βG′3 fraction was similar in reducing and nonreducing conditions, with lower intensity vicilin, Lα, Lβ, and βlg bands and the absence of a J


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AUTHOR INFORMATION

Corresponding Author

*(R.S.) E-mail: [email protected]. Phone: +33 380 774 051. Fax: +33 380 774 047. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Roquette Group SA (Lestrem, France) and Ingredia Group SA (Arras, France) for providing the raw material.

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ABBREVIATIONS USED ANS, 1-anilinonaphthalene-8-sulfonic acid; βlg, beta-lactoglobulin; BSA, bovine serum albumin; CV, pea convicilin 7S; Cas, casein; DF, diafiltration; DSC, differential scanning calorimetry; DLS, dynamic light scattering; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); DTT, dithiothreitol; Dh, hydrodynamic diameter; Ev, elution volume; Glob, globulin; H0, surface hydrophobicity; I, band intensity; Lαβ, legumin subunit; Lα, acidic legumin polypeptide; Lβ1-2, basic legumin polypeptide; Leg, pea legumin 11S; Lox, lipoxygenase; Mw, molecular weight; NAC, N-acetylcysteine; NC, native conditions; NR, nonreducing conditions; pHi, isoelectric pH; R, reducing conditions; SEC-HPLC, size exclusion chromatography; S−/ S−S, sulfhydryl/disulfide bond; SH, thiol group; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; Td, temperature of denaturation; UF, ultrafiltration; V, pea vicilin 7S; WPI, whey protein isolate; wt, weight; ΔHd, enthalpy of denaturation

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