Characterisation and foaming properties of hydrolysates derived from rapeseed isolate

Colloids and Surfaces B: Biointerfaces 49 (2006) 40–48

Characterisation and foaming properties of hydrolysates derived from rapeseed isolate C. Larr´e a,∗ , W. Mulder b , R. S´anchez-Vioque a , J. Lazko a , S. B´erot a , J. Gu´eguen a , Y. Popineau a a

INRA, Unit´e de Recherche sur les Prot´eines V´eg´etales et leurs Interactions, BP 71627, 44316 Nantes Cedex 3, France b ATO-DLO, P.O. Box 17, NL-6700 AA Wageningen, The Netherlands Received 1 February 2005; accepted 4 February 2006 Available online 6 March 2006

Abstract Two hydrolysis methods used to obtain rapeseed isolate derivates were compared: chemical hydrolysis performed under alkaline conditions and pepsic proteolysis performed under acidic conditions. The mean molecular weights obtained for the hydrolysates varied from 26 to 2.5 kDa, depending on the level of hydrolysis. Further characterisation showed that, at the same level of hydrolysis, the chemical hydrolysates differed by their charges and hydrophobicity from those derived from enzymatic digestion. Analysis of the foaming properties showed, for both cases, that a limited degree of hydrolysis, around 3%, was sufficient to optimise the foaming properties of the isolate despite the different physicochemical properties of the peptides generated. The study of foaming properties at basic, neutral and acidic pHs showed that the hydrolysate solutions yielded dense foams which drained slowly and which maintained a very stable volume under the three pH conditions tested. © 2006 Elsevier B.V. All rights reserved. Keywords: Rapeseed isolate; Alkaline hydrolysis; Pepsin; Proteolysis; Foaming properties

1. Introduction Most of the rapeseed production that undergoes the industrial deoiling process results in large quantities of rapemeal enriched in proteins (40–50%), but with a limited protein solubility. It is only used in animal feed at this time, whereas it could provide valuable raw material for non-food applications as a result of a number of physical, chemical or enzymatic modifications. Numerous modified proteins exhibit improved functionalities such as solubility, emulsification and foaming [1–6] that could be used in non-food applications such as paint, coating and adhesive formulations. These properties are linked to the ability of the proteins to stabilise oil:water or air:water interfaces. The way that proteins adapt to various surfaces and interfaces is of great importance to their ability to be used in foaming systems. Protein solubility is a major prerequisite for foaming properties but foam formation and stability are closely related to the

∗

Corresponding author. Fax: +33 02 4067502. E-mail address: [email protected] (C. Larr´e).

0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.02.009

adsorption rate at the interface and to the structure of the proteins. Enzymatic or chemical hydrolysis of proteins has been used to improve their solubility and to provide them with new functional properties. These modifications generate polypeptides and peptides that differ from the initial proteins by their molecular size, number of ionisable groups and surface hydrophobicity. This induces foaming properties different from those of the initial substrate. As compared to proteins, peptides diffuse more rapidly to the interfaces. However, a minimal molecular weight is required to express surface properties and to stabilise foams. Foaming properties of milk proteins can be improved through proteolysis [7]. Van der Ven et al. showed that molecular weight distribution of whey hydrolysate was correlated to foam-forming properties and, in the case of casein hydrolysates, to foam stability [8]. Studying casein hydrolysate, Caessens et al. [9] showed that the most hydrophobic peptides presented the best surfaceactive properties but only when they were not charged. In the case of ␤ lactoglobulin, proteolysis improved foam overrun and a limited hydrolysis resulted in an increased affinity for the interface [10,11]. The solubility and the foaming properties of wheat

C. Larr´e et al. / Colloids and Surfaces B: Biointerfaces 49 (2006) 40–48

gluten, which is water-insoluble, were greatly increased using proteolysis [12,13]. The foaming properties were improved for all gluten hydrolysates until an optimal degree of hydrolysis was reached. Beyond this point, the foam-stabilising properties decreased. Moreover, Popineau et al. [14] showed that the hydrophobic fraction of this hydrolysate considerably enhanced the stability of the foam. Alkali treatments are also very effective for solubilising proteins as a result of peptide and other amide bond hydrolysis [15]. They have been used in the processing of industrial proteins, often during the initial extraction stages in the case of meals, and the generated products were further modified for various applications [16]. Only a few studies have focused on the physicochemical or foaming properties of peptides resulting from alkaline hydrolysis [17]. Cruciferin (12S globulin) and napin (2S albumin) are the major proteins found in rapeseed isolate. Cruciferin has a molecular mass of 300 kDa and is organised in a hexameric structure. Each of the six subunits is composed of two polypeptidic chains (α and β) of about 30 and 20 kDa, linked by a disulfide bond [18]. Napin is smaller, with a molecular weight between 12 and 14 kDa. It is composed of two polypeptides with a molecular mass of approximately 4 and 9 kDa, linked by two interchain disulfide bonds; its compact structure is stabilised by two additional intrachain disulphide bridges [19]. Krause and Schwenke [20] showed that the interfacial activity of an isolate produced at lab scale differs from that of its two constitutive proteins. Moreover, its adsorption properties were also different from the properties of a mixture with the same ratio of cruciferin and napin [20]. Nitecka et al. [21] showed that native isolated napin presented excellent foaming properties and Vioque et al. [22] reported that hydrolysis by alcalase was a good way to enhance rapeseed isolate foaming and emulsifying properties. They showed an optimum for low degrees of hydrolysis. As part of a large cooperative European research programme (ENHANCE: Green chemicals and biopolymers from rapeseed meal with enhanced end-performances), aimed at enlarging the application field of rapeseed components, oil extraction process were adapted to avoid the denaturation of the protein fraction of the grain. A process for the production of isolate from this mildly deoiled meal was developed [23]. This isolate was characterized by a high solubility and very good emulsifying properties [24]. Because the foaming properties were limited, this study is focused on the improvement of the foaming properties of the isolates through the use of controlled hydrolysis. Foams are widely used in food and non-food applications in the pharmaceutical, engineering and acoustic industries, among others, in which enzymatically or chemically modified proteins could effectively be used. The isolate used in this study was produced at the pilot scale and was further subjected to hydrolysis. Enzymatic modifications using pepsin and chemical modifications using alkaline hydrolysis were compared. The hydrolysates were characterised and their foam-forming and foam-stabilising properties were investigated using a sparging test. Finally, we characterised the peptidic fractions adsorbed at the air–water interface.

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2. Materials and methods 2.1. Material Rapeseed isolate was obtained from the IVV Fraunhofer Institute (Freising, Germany). The soluble aqueous fraction obtained after suspension of mildly deoiled meal, produced by CETIOM (Bordeaux, France) from dehulled rapeseed, was subjected to successive precipitations and finally to ultrafiltration fractionation. The isolate used in this study corresponds to the filtrated protein II described by W¨asche and Sch¨onweitz [23]. Its protein content was 96 ± 1% d.m. and its solubility at pH 7 was 93 ± 0.4% [24]. Napin and cruciferin were purified according to B´erot et al. [25]. Casein from cow milk was purchased from Sigma–Aldrich, Lyon, France. A commercial pea protein isolate was obtained from Provital (Warcoing, Belgique). 2.2. Alkaline hydrolysis A 10% (w/w) dispersion of rapeseed protein isolate was prepared in 0.25, 0.50, 1.50, 2.00, 2.50, 3.00 and 4.00 M sodium hydroxide. The alkaline protein suspension was stirred for 24 h at 20 ◦ C. The reaction was stopped by neutralising the solution to pH 7 with HCl. The salt formed during neutralisation was finally removed by nanofiltration. 2.3. Enzymatic hydrolysis The isolate was dispersed in water at 50 mg/mL and then adjusted to pH 4.0 with hydrochloric acid. Hydrolysis was performed at 37 ◦ C under stirring using a pepsin for various times; the peptide/substrate ratio (E/S) was 1/50. Enzyme inactivation was achieved by heating the dispersion at 90 ◦ C for 5 min in a water-bath. The hydrolysates were neutralised by the addition of ammonia prior to freeze-drying. 2.4. Hydrolysate characterisation The amount of free amino groups was determined by the OPA1 method of Frisher et al. [26], according to Linares et al. [27]. The number of amino groups was determined with reference to a standard curve obtained with l-leucine. The degree of hydrolysis is defined as the percentage of cleaved peptidic bonds. The value given for each hydrolysate is the mean of three determinations. The number of terminal carboxylic groups was determined by titration of a 0.3 g sample in demineralised water with pH values ranging from 6 to 3. 2.5. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) Electrophoresis was performed using 15% acrylamide separation gels and 6% acrylamide stacking gels under denaturing and reducing conditions. Reduction was achieved by heating 1

Orthophthaldialdehyde.

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the sample in boiling water for 5 min in the presence of 2mercaptoethanol (5%, v/v). 2.6. Gel filtration The molecular distribution was determined by size exclusion chromatography on a Superdex 75 column (10 mm × 300 mm, Amersham Biosciences) eluted with 0.05 M phosphate buffer pH 8.2, 0.20 M NaCl, 0.5% SDS2 for isolates and total hydrolysates. The samples were solubilised in the eluting buffer and centrifuged at 10,000 × g for 10 min. A Superdex peptide column (Amersham Biosciences) was used for napin and its hydrolysates after equilibration with 0.02 M Tris–HCl buffer, pH 8.0. The supernatant was then filtered through a 0.2 ␮m membrane before 100 ␮L were loaded onto the column. The elution was performed at 0.5 mL/min and the detection at 220 or 280 nm. 2.7. RP-HPLC The isolate, hydrolysates and peptide fractions recovered from foams (see below) were analysed by RP-HPLC with a C18 ˚ 5 ␮m, 4.6 mm × 250 mm, Nucleosil, Macherycolumn (300 A, Nagel). Proteins or peptides (1 mg/mL) were solubilised in 0.01% TFA and 100 ␮L were loaded onto the column. They were then eluted by a linear gradient of acetonitrile concentration (0–80% in 40 min). Column temperature was maintained at 50 ◦ C in an oven. Peptide detection was performed at 220 nm. Since the chromatograms presented a bimodal shape, two groups were considered: the first group eluted was referred to as ‘hydrophilic’ and the second group as ‘hydrophobic’. Their relative proportions were calculated on the basis of their surface area.

under three pH conditions: 0.1 M phosphate buffer, pH 7, 0.1 M citrate buffer, pH 3, and 0.1 M Tris buffer, pH 8.5. Protein solutions at 1 mg/mL were placed in the sparging chamber at the base of a glass column. Foam was generated by sparging nitrogen through a porous glass disk at a rate of 25 mL/min (pressure: 20 MPa) into 8 mL of the protein solution until a foam volume of 35 mL was reached. The volume of initial or residual liquid under the foam was measured by conductivity through two electrodes located in the sparging chamber. Conductivity measurements, as a function of time (Ct ) and with reference to the conductivity of the buffered test solution (Cinit ), were used to calculate the volume of liquid in the foam (VL ): VL = Vinit [1 − (Ct /Cinit )], where Vinit is the volume of sample solution (8 mL) introduced into the sparging chamber. Foaming capacity was estimated from the sparging time required to form 35 mL of foam and from the maximal density of foam Dmax (with Dmax = maximum volume of liquid in the foam/maximum volume of foam). Foam volume was monitored for 20 min after the bubbling was stopped, using a video camera. The stability was estimated from the time of half-drainage (t1/2 ) and from the residual fraction of foam volume after 20 min (final foam volume/maximal foam volume, %). The measurements were performed in duplicate. 2.9. Characterisation of the peptides adsorbed in the foam Foams were prepared from hydrolysate solutions at 3 mg/mL and the remaining foam was recovered after complete drainage has occurred. This drained foam was then broken up by centrifugation to recover the interfacial liquid. The peptidic composition of the interfacial liquid fraction was analysed by RP-HPLC on a C18 column as described above.

2.8. Functional properties

3. Results and discussion

2.8.1. Surface activity Surface tension measurements were performed using a tensiometer (Kr¨uss, Palaiseau, France) equipped with a platinum Wilhelmy plate. After checking the surface tension of the buffer solution at 72.9 ± 0.2 mN, 100 ␮L of the protein solution at 25 mg/mL in 0.1 M sodium phosphate buffer, pH 7.0, were injected into the subphase. The final peptide concentration in the solution was 50 ␮g/mL. The surface tension was continuously recorded over several hours at 20 ◦ C until a constant value was observed. The surface pressure at equilibrium was then calculated as πe = γ 0 − γ e , where γ 0 and γ e are the surface tensions at t = 0 and at equilibrium, respectively. All measurements were made in triplicate.

3.1. Characterisation of hydrolysates

2.8.2. Foaming properties Foam formation and stability were analysed with the Foamscan instrument (IT Concept, Longessaigne, France) as described by Sarker et al. [28] with small modifications. Foaming properties of protein and peptide solutions were determined 2

Sodiumdodecylsulphate.

Hydrolysates obtained by enzymatic or chemical means were characterised by their degree of hydrolysis, their molecular weight distribution and their hydrophobicity profile. 3.1.1. Enzymatic hydrolysates The isolate was gradually degraded by pepsin. The degree of hydrolysis, estimated from the amount of free amino groups released, reached 12% after 24H. Electrophoresis revealed that the largest peptides, corresponding to the cruciferin subunits, were cleaved at the very beginning of the reaction. When DH was between 2 and 3%, the molecular weights of the peptides were below 20 kDa, but some faint bands were observed at molecular weights up to 60 kDa (Fig. 1A). At 4% DH, the peptides were characterised by a broad range of molecular weights lower than 15 kDa. These fragments were further degraded when hydrolysis was continued (not shown). These mass estimations were confirmed by gel filtration. In the case of the hydrolysate with a DH of 3%, the chromatogram revealed a main peak representing material with a mean molecular weight of 15 kDa. The absence of polypeptides with a higher molecular weight indi-

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Fig. 1. SDS electrophoresis of hydrolysates under reducing conditions: (A) pepsic hydrolysates at various degrees of hydrolysis (%); (B) hydrolysates obtained at various sodium hydroxide concentrations (M).

cates that both the acidic and basic polypeptides of the cruciferin were degraded, although this method was not resolutive enough for napin polypeptides. An extra gel filtration analysis was performed on an hydrolysate of napin obtained under the same conditions (Fig. 2). Hydrolysis induced only a slight decrease of the peak corresponding to the native form of napin. These samples of native and hydrolysed napin were further reduced and analysed by gel filtration (Fig. 2). The profile of the reduced napin showed two peaks corresponding respectively to the large and the small subunits. These two peaks are still present in the hydrolysate pattern, showing that hydrolysis was only partial. However, a new peak and a shoulder of MW higher than the small subunit were observed too, in conjunction with a decrease of the area of the large subunit peak. So, these peptides derived from the large subunits but were released only after reduction of SS bonds. This means that the enzymatic cleavage sites were located between two cysteins engaged in disulfide bonds. After hydrolysis these fragments remained bound together, unless disulfide bonds were broken.

Fig. 3. RP-HPLC characterisation of (—) purified napin and ( ciferin.

) purified cru-

The RP-HPLC profile of purified napin and cruciferin are shown in Fig. 3. The profile of the native isolate showed three main peak groups (Fig. 4). By comparison with patterns of pure napin and cruciferin (Fig. 3), the first group corresponded to the 2S albumins (napin); the two others corresponded to the cruciferin polypeptides. The resolution of the RP-HPLC pro-

Fig. 2. Gel filtration of native (—) and hydrolysed napin with a DH of 2.9% (

) under non-reducing conditions (A) and reducing conditions (B).

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C. Larr´e et al. / Colloids and Surfaces B: Biointerfaces 49 (2006) 40–48 Table 1 Mean molecular weight estimated by gel filtration, degree of hydrolysis and level of deamidation of hydrolysis obtained using different concentrations of NaOH

Fig. 4. RP-HPLC characterisation of the native isolate (—), the 1.5 M NaOH hydrolysate (
) and the pepsic hydrolysate DH 3% (䊉).

file of the hydrolysate with a DH of 3% was lower but showed two broad peaks eluted between 20 and 40% acetonitrile and from 40 to 61% acetonitrile. No peptide was as hydrophobic as the native cruciferin components. The 12S polypeptides disappeared completely, whereas a broad peak overlaid the initial peak of native napin (Fig. 4). According to the analysis performed in electrophoresis and gel filtration on pure napin, some non-hydrolysed napin could be present under this broad peak. This once again showed that 12S polypeptides were degraded by pepsin and confirmed the preservation of the napin under non-reducing conditions. The proteolysis with pepsin was carried out at pH 4. These acidic conditions are known to induce a partial dissociation of the globular structure of the 12S globulins accompanied by a partial denaturation of the subunits [20]. This opening of the structure increased the exposure of cleavable sites, especially on the ␤ polypeptides, which are known to be located in the inner part of the native cruciferin. Proteolysis assays on reduced napin confirmed that the low degradation of the native form is due to a low accessibility of the cleavage sites (results not shown). 3.1.2. Chemical hydrolysates Hydrolysis under alkaline conditions was studied using sodium hydroxide concentrations varying from 0.25 to 4 M.

NaOH (M)

MW

% DH

% Deamidated residues

0 0.25 1.50 2.00 4.00

43689 40138 26278 6965 2545

0 0.5 2.3 2.9 5.8

0 9 72 76 86

The reactions were performed for 24H at room temperature. Higher temperatures, which would accelerate the hydrolysis, were not suitable due to the rapid denaturation and precipitation of rapeseed protein. The extent of protein hydrolysis by alkaline processing was dependent on the sodium hydroxide concentration and the amount of hydrolysed peptide bonds varied from 0.5 to 6%. This low degree of cleavage reflected the stability of peptide bonds involved in hydroxide ion-catalysed reactions and, under the experimental conditions, a 1 M sodium hydroxide concentration was required to obtain a significant modification. The extent of deamidation was calculated on the basis of the amount of amino and carboxylic acid groups which were formed by the hydrolysis of asparaginyl and glutaminyl side chains (Table 1). High levels of deamidation were reached with NaOH concentrations greater than 1 M. This means these hydrolysates bear a large number of negative charges. As shown in Fig. 1B, only one main broad zone was observed in SDS-PAGE, corresponding to molecular weights lower than 14 kDa for hydrolysates treated with 1 M NaOH and higher concentrations. The blurred appearance of the bands was probably due to the non-specificity of the alkaline cleavage and to the hydrolysis of the unsubstituted amide bonds that occurs even under mild alkaline conditions [15]. The analysis of the molecular weight distribution of these hydrolysates by gel filtration confirmed a very limited hydrolysis with 0.2 M NaOH. For the higher NaOH concentrations, broad distributions of the peptide sizes yielded mean molecular weights varying from 26 kDa (1.5 M NaOH) to 2.5 kDa (4 M NaOH). The comparison of SDS-PAGE and gel filtration results indicated that the large polypeptides (over 26 kDa) obtained by the mildest alcaline conditions were most probably composed of several peptides linked by disulfide bonds, because under reducing conditions no peptide exhibited a MW higher than 20 kDa. The hydrophobic profile of the peptides obtained by chemical hydrolysis with 1.5 M NaOH displayed two main broad peaks (Fig. 4). The first one, composed of the more hydrophilic peptides (CP1), was eluted with a percentage of acetonitrile lower than 61%, in the same concentration range as the native napin, while the second one composed of more hydrophobic peptides (CP2) eluted from 61 to 75% acetonitrile as the components of native cruciferin. For similar degrees of hydrolysis, around 3%, the chemical hydrolysate contained a higher proportion of hydrophobic peptides than the enzymatic hydrolysate for which 95% of peptides were eluted with a percentage of acetonitrile lower than 61% (Fig. 4).

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Fig. 5. Adsorbtion kinetics of the isolate (×), purified cruciferin (–), purified napin (- -), 3% DH pepsic hydrolysate () and 1.5 M NaOH hydrolysate (♦).

3.2. Surface tension measurements The effect of hydrolysis on the interfacial behaviour of rapemeal products was studied using the Wilhelmy plate technique. The surface pressure modifications measured at pH 7 with pepsic hydrolysate and one alkaline hydrolysate with the same DH (around 3%) were compared to those of native isolate, purified 12S and 2S (Fig. 5). For all samples, the plateau value Π e was reached after 1 h. The surface tension measured for purified 12S showed a lag phase before decreasing. This delay could be linked to the slower diffusion of the globulin to the interface in relation to its large molecular size. The decrease of surface pressure was immediate with the 2S napin (smaller than 12S), although the equilibrium pressure was higher. Napin appeared less effective for decreasing the surface tension than the 12S polypeptides as previously reported [20]. Napin surface hydrophobicity as estimated from RP-HPLC was lower as well. The behaviour of the native isolate was very similar to that of the purified 12S globulin (lag phase, equilibrium pressure), suggesting that the 12S globulins determine its surface properties. Because of the lower molecular weight of peptides, the initial surface tension decreased more quickly with both types of hydrolysates than in the case of the native isolate. The chemical hydrolysate showed the sharpest decrease of the initial surface tension and the lowest surface tension. The filtrated isolate showed an equilibrium surface pressure of 21.2 mN/m, which was significantly lower than the value of 31.8 mN/m for a rapeseed protein isolate prepared at a laboratory

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scale, determined by Krause and Schwenke [20]. This difference was probably due to the preparation of the isolates. On the other hand, the values found for purified cruciferin (22.2 mN/m) and napin (14.7 mN/m) were in agreement with those previously reported by Krause and Schwenke. Cruciferin represents almost 60% of the rapeseed storage proteins. Accordingly, the equilibrium pressure of the native isolate was close to that found for cruciferin. This indicated that the interfacial behaviour of the isolate was dominated by that of cruciferin. The equilibrium surface pressures that were measured for the enzymatic hydrolysates were lower than that of the isolate (14.7 mN/m for the hydrolysate with a 3% DH) but similar to that measured for napin. This could be explained by the lower surface hydrophobicity of the hydrolysate due to the enzymatic cleavage of the 12S polypeptides as shown by RP-HPLC (Fig. 3) and to the resistance to pepsic hydrolysis of napin. The chemical hydrolysates prepared with 1.5, 2, 2.5 and 3 M NaOH resulted in Π e values of 23.5, 24.4, 24.0 and 23.7 mN/m, respectively. They were very comparable to the Π e of 12S, which did not reveal a strong influence of the DH on the equilibrium pressure. The differences between surface behaviour of the enzymatic and the chemical hydrolysates could be explained by higher amounts of negative charges caused by deamidation and the presence of more hydrophobic peptides in the chemical hydrolysates. 3.3. Foaming properties 3.3.1. Effect of the degree of hydrolysis on the foaming properties measured at pH 7 3.3.1.1. Foam formation. The sparging time required to reach a final foam volume of 35 mL was used as a measure for foamforming ability. Compared to the native filtrated isolate, the sparging time required for all the tested hydrolysates was slightly reduced (Table 2). This indicated that all the hydrolysates possessed a very good ability to generate foam that was even more pronounced for the enzymatic hydrolysates than for the chemically produced hydrolysates. Regardless of the type of treatment, the degree of hydrolysis did not influence the sparging time. The ability to incorporate liquid in the foam was measured by the maximal density. This is also an estimation of the potential foam expansion obtainable with whipping foam-

Table 2 Foaming properties of rapeseed isolate, enzymatic and chemical hydrolysates Sparging time (s)

Maximal density

Half drainage time (s)

Final foam volume (%)

Casein Pea isolate Filtrated isolate

72 69 71

0.131 0.103 0.142

176 178 125

96 94 49

Pepsic hydrolysate DH 3% DH 7%

65 64

0.169 0.166

287 273

91 97

Chemical hydrolysate 1.5 M (DH 2.3%) 2 M (DH 2.9%) 2.5 M (DH 3.2%) 3 M DH 4.1%)

68 69 69 69

0.173 0.176 0.172 0.163

306 217 219 213

95 92 94 94

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ing processes, since a direct correlation exists between foam density with bubbling and foam overrun with whipping [29,30]. All hydrolysates exhibited a higher foam density than the native filtrated rapeseed isolate. However, the densities of the foams prepared with chemically modified hydrolysates with DH lower than 3% were higher than those of the enzymatic hydrolysates. Clearly, a limited hydrolysis was sufficient to improve the foaming ability of the rapeseed isolate. When compared to casein or pea protein isolate, which are proteins widely used for their functional properties in food applications, the hydrolysates showed a much denser foam, corresponding to a better ability to foam. 3.3.1.2. Foam stability. The ageing of the foam was analysed by recording the drainage kinetics and the evolution of the foam volume. Whereas the foam made with the filtrated isolate broke up rapidly within the first 200 s after bubbling, the volumes of the foams prepared with any of the hydrolysates remained unchanged during the analysis time. The decrease of the foam volume was less than 10% of the initial volume after 20 min (Table 2). The drainage, which is the flow of liquid out of the foam due to gravity, was slower for the hydrolysates than for the filtrated isolate. This is reflected by values of the half drainage time (t1/2 ), which were at least 1.7 times longer for the hydrolysates. This indicated that the peptides and polypeptides anchored at the interface were able to interact and form more hydrated films. The slowest drainage was obtained for the two enzymatic hydrolysates and the 1.5 M NaOH hydrolysate. Increasing the DH of the chemical hydrolysates tended to accelerate the drainage but the foam volume stability was not altered. This acceleration of drainage was linked to the peptide size decrease but not to charges, because the number of deamidated residue of the 2.0 M NaOH hydrolysate was the same as for the 1.5 M NaOH hydrolysate. Based on these results, the best foaming properties were obtained with the enzymatic hydrolysate with a 3% DH and the chemical hydrolysate with a 2.3% DH (1.5 M NaOH). In both cases, a limited hydrolysis seemed to be more effective for enhancing foaming properties but the pepsic hydrolysate DH 7% still presented convenient foaming properties. This result is in agreement with Vioque et al. [22] who found that alcalase

hydrolysates with DH from 3.1 to 7.7% showed good foaming properties at an optimum with DH 3.1%. Chemical hydrolysates also presented good foaming properties with an optimum at DH 2.3%. For higher DHs the drainage time decreased. 3.3.1.3. Relationships with surface activity of isolate and hydrolysates. The values of Π e were not simply related to the foaming properties in the foam formation step nor with the foam stability. The main feature explaining the higher foaming properties of the hydrolysates was the rate of migration to the air–water interface of the peptides when compared to intact proteins, as seen in the first part of the adsorption kinetics. For peptides, no lag phase was observed and the surface tension decreased rapidly, whereas this decrease was slower for the cruciferin and a lag phase of several minutes was found with the isolate. This means that the peptides were able to migrate rapidly to the bubble surface during the foaming process, thus, stabilize very small and homogeneous bubbles. The isolate proteins (mostly cruciferin) migrated slowly and adsorbed at the surface of larger (and less homogeneous) gas bubbles. This resulted in the higher density of the peptide foams. Foam stability was probably determined by initial foam texture (homogeneous versus heterogeneous bubble sizes) and the mechanical properties of the interfacial film rather than by the equilibrium pressure, although the high Π e of the chemical hydrolysate could contribute to the highest stability of this foam. 3.3.2. Effect of pH on foaming properties The behaviour of the enzymatic hydrolysate (DH 3%) and the alcaline hydrolysate (DH 2.3%), showing the best properties at pH 7, were compared at two other pHs. 3.3.2.1. Foam formation. The sparging time was only minimally affected by the pH of the protein or peptide solutions (Fig. 6A). For casein, pea and rapeseed isolates, a slightly longer time was observed at pH 8.5. No difference was found for the enzymatic (DH 3%) and chemical hydrolysates with a 2.3% DH. The pH had a much greater impact on the maximum density of the foam of pea and rapeseed products (no change for casein) (Fig. 6B).

Fig. 6. Effect of the pH on the foam formation parameters: (A) sparging time; (B) maximum density of foam. FI: filtrated isolate, EH: enzymatic hydrolysate DH 3%, CH: chemical hydrolysate DH 2.3%. (
) pH 3; ( ) pH 7; ( ) pH 8.5.

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47

Fig. 7. Effect of the pH on the foam stability parameters: (A) half drainage time; (B) final foam volume/maximum foam volume, %. FI: filtrated isolate, EH: enzymatic hydrolysate, CH: chemical hydrolysate. (
) pH 3; ( ) pH 7; ( ) pH 8.5.

The density of foams prepared with pea isolate, rapeseed isolate and the enzymatic hydrolysate was reduced at pH 8.5. For the chemical hydrolysate, the density was maximum at pH 7. Nevertheless, at each pH, the foaming ability of the hydrolysates remained higher than that of the filtrated isolate and at least similar to casein. 3.3.2.2. Foam stability. The drainage rate of the foams (reported here by the half-drainage time) was influenced by pH (Fig. 7A). The drainage of casein and pea isolate foams was the slowest at pH 7 (longest half-drainage time). The drainage of the foam prepared with the filtrated isolate was very slow at pH 3 but rapid at pH 7 and 8.5. The variation of drainage rates of the foams made with the hydrolysates was limited at pH 3 or 7 but the half-drainage time considerably decreased at pH 8.5. At this pH, the hydrolysis improves the foaming properties as shown by the increase of the half time drainage and the final foam volume compared to those of filtrated isolates. The stability of the foam volume was improved at pH 7 and 8.5 by the partial enzymatic hydrolysis of the rapeseed isolate (Fig. 7B). Although the foam made with the pea isolate was relatively dry (low maximum density, short half-drainage time), its structure was very stable since the foam volume was stable during ageing and did not depend on pH. This pH-unaffected stability was also a characteristic of the foams made with the chemical hydrolysate while the foams prepared with the enzymatic hydrolysate at DH 3% were less stable at acidic pH. Moreover, the foaming properties of the hydrolysates were less altered by the pH than those of the native proteins.

filtrated isolate was enriched in 2S albumins. This preferential adsorption has to be related to the faster diffusion to interface of the smaller napin shown above. The dominating influence of the rapeseed albumin was previously observed at pH 8 by Krause and Schwenke [20]. Based on the results of the surface tension measurements, the improved foaming properties of the isolate at pH 3 suggest that the dissociation of 12S hexamers facilitates the adsorption of the cruciferin subunits at the expense of the napin. In the case of peptides produced by alkaline hydrolysis, the hydrophobic profiles of the adsorbed fractions were very different from the initial solution composition. The interfacial liquid was enriched in the more hydrophobic peptides (CP2), as illustrated in the case of the hydrolysate prepared with 1.5 M NaOH (Fig. 9). The different fractions of foams prepared with all of the hydrolysates were analysed by RP-HPLC and the proportions of CP1, CP2 defined in Section 3.1. were calculated (Fig. 10). In the case of the enzymatic hydrolysate with a 3% DH all the peptides were adsorbed at the same rate and no difference was observed on the chromatography patterns of the drained and interfacial liquid. As shown in Fig. 4, the peptides generated by pepsin hydrolysis are less hydrophobic than the chemical peptides preferentially adsorbed. The preferential adsorption of the CP2 fraction of the chemical hydrolysates is probably related to the combination of small size, in agreement with the very fast

3.4. Characterisation of the peptides adsorbed at the air–water interface The rapeseed protein hydrolysates yielded more stable foams than the filtrated isolate. We therefore attempted to characterise the proteins and peptides anchored at the air:water interface in the bubbles of the foams prepared at pH 7. The composition of the remaining interfacial liquid around the bubbles after complete drainage was compared to that of the whole hydrolysate. The drained foams were collected and broken up by centrifugation. The interfacial liquid was then recovered and analysed by RP-HPLC (Fig. 8). The interfacial liquid from foam made of

Fig. 8. RP-HPLC characterisation of the different fractions of a foam prepared with the native isolate: ( ) initial isolate solution and (—) interfacial liquid of the remaining foam after 24 h.

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C. Larr´e et al. / Colloids and Surfaces B: Biointerfaces 49 (2006) 40–48

Acknowledgements This research was funded by the European Community within the frame of ENHANCE QLK5 1999-01442, coordinated by Jacques Gu´eguen (INRA Nantes, France). References

Fig. 9. RP-HPLC characterisation of the different fractions of a foam prepared with the 1.5 M NaOH hydrolysate: (
) initial hydrolysate solution, (—) drained liquid and ( ) interfacial liquid of the remaining foam after 24 h.

Fig. 10. Proportion of hydrophobic peptides (CP2) for chemical hydrolysates: in the initial liquid (
) and in the interfacial liquid of the foam (). These results are the mean of three measurements.

decrease of the surface tension and a high hydrophibicity, in line with the lowest equilibrium surface tension (Fig. 5). 4. Conclusion Chemical as well as enzymatic hydrolyses were successfully used to improve the foaming properties of a filtrated rapeseed isolate. Only a limited hydrolysis was required since the properties did not further improve beyond a DH of about 3%. The chemical hydrolysates differed from the enzymatic hydrolysates by their number of negative charges resulting from deamidation occurring at the same time as peptide bond cleavage, as well as by a higher concentration of hydrophobic peptides. The ability of the chemical hydrolysates to stabilise the foam bubbles was mainly due to the hydrophobic peptides that were preferentially adsorbed at the air:water interface. However, both types of hydrolysates showed similar foaming properties, whereas the best performances were obtained at acid and neutral pHs. Moreover, their properties were superior to those of other industrially produced proteins. Therefore, they could easily be used in various formulations for either food or non-food applications.

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