Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin

Food Hydrocolloids 27 (2012) 154e160

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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Effect of pulsed-light processing on the surface and foaming properties of b-lactoglobulin Estibalitz Fernández a, Mari Luz Artiguez a, Iñigo Martínez de Marañón a, Maider Villate b, Francisco J Blanco b, c, Juan-Carlos Arboleya a, * a b c

AZTI-Tecnalia Food Research Institute, Parque Tecnológico de Bizkaia, Astondo Bidea, Edificio 609, 48160 Derio, Bizkaia, Spain CIC-BIOGUNE, Parque Tecnológico de Bizkaia, Edificio 801 A, 48160 Derio, Bizkaia, Spain IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 March 2011 Accepted 1 August 2011

Pulsed-light processing was used to treat b-lactoglobulin (BLG) solutions. The impact of pulsed light (PL) on the structural properties of this protein was explored through far-UV, CD spectral analysis, size exclusion chromatography, surface hydrophobicity and NMR spectroscopy. Changes on these physicochemical properties were related to surface rheology, surface tension, foam stability and foam capacity of the non-treated and treated BLG to elucidate adsorption mechanism and consequences on foaming capacity. Conformational modification of BLG was related with PL total fluence as important conformational changes increased when total fluence was higher. Consequently, adsorption rate of treated BLG at the air/water interface was faster than native BLG. Additionally, treated BLG formed highly elastic interfaces. This was found to have an impact on the foam stability. Pulsed-light treatment seemed to enhance the overall strength of the interface, resulting in more stable foams. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Pulsed-light process b-Lactoglobulin Surface properties Foaming properties

1. Introduction Bubbles are desirable elements in a wide range of applications, particularly in food, since consumer perception of quality is strongly influenced by appearance. Aerated systems are however thermodynamically unstable and in a fluid system will eventually break down (Dickinson & Wasan, 1997). Therefore, the role of surface active ingredients like proteins is crucial for the formation and stability of food foams. Whey proteins are currently the most used food proteins in foodstuffs because of their excellent functional properties. Great effort has been made during the last years to improve those functional properties by applying different treatments, including chemical (Enomoto et al., 2007; Wooster & Augustin, 2007), physical (Phillips, Schulman, & Kinsella, 1990; Yang, Dunker, Powers, Clark, & Swanson, 2001) or enzymatic treatments (Davis, Doucet, & Foegeding, 2005). The effects of the applied treatment in the structural properties of whey proteins have been extensively studied, especially for b-lactoglobulin (BLG), mainly because all these treatments may have an impact in protein structure, modifying the kinetics of protein adsorption at the interface, the time needed for the protein to rearrange upon

* Corresponding author. Tel.: þ34 946 574 000; fax: þ34 946 572 555. E-mail address: [email protected] (J.-C. Arboleya). 0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.08.001

adsorption at the interface and the ability to interact with adjoining proteins (Croguennec, Renault, Bouhallab, & Pezennec, 2006). Proteins that can be altered to adsorb more rapidly, and produce stronger interfaces are generally capable of producing finer and more stable foams. The drawback of this is that some treatments can cause aggregation as interactions between proteins in the bulk are higher. This provokes a reduction in protein solubility and thus, in protein availability to be adsorbed and consequently results in poor foam production. On the other hand, the dairy industry produces significant amounts of liquid wastes, mostly whey obtained during the cheese making process. This high-content-protein source must be treated for microorganism elimination by specific treatments while minimizing protein denaturation. Pasteurization has been used to decontaminate whey. However, high temperature processes are known to denature whey proteins (Anema & Li, 2003) causing substantial changes in their nutritional, organoleptic or technological properties. For this reason, an important challenge would be to develop non-thermal technologies which can prevent adverse thermal effects and produce safe food products (Barbosa-Cánovas, Góngora-Nieto, & Swanson, 1998). Among others, pulsed-light (PL) process consists of a successive repetition of short duration and high power pulses of broadband emission light (200e1000 nm) with a considerable amount of light

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in the short-wave UV spectrum (Wekhof, 2000). Pulsed-light process has been shown to be effective in inactivating a wide broad of microorganisms involved in food products spoilage (Lasagabaster, Arboleya, & Martínez de Marañón, 2011; Rowan et al., 1999). However, there is no research about the impact of pulsed-light processing in the functional properties of food proteins. Only Elmnasser et al. (2008) studied the effect of PL treatment in milk proteins and lipids concluding that minor changes in protein structure were found, despite of changes in the polarity of the tryptophanyl residue microenvironment of BLG solutions and changes in the tryptophan indole structure and some protein aggregation. Therefore, the aim of this research is to study the conformational changes in BLG induced by applying a PL treatment and how these changes affect their surface properties and hence, their foaming properties. 2. Materials and methods 2.1. Materials

b-Lactoglobulin (BLG; L-2506 approximately 80% purity), derived from bovine milk, were purchased from Sigma chemicals (Gillingham, UK). Solutions were prepared at different concentrations in ultrapure water (g0 ¼ 72.6 mN m1 at 20  C), allowed to equilibrate for 60 min, and used without further purification. Initial pH was 6.8 and it did not change after treatments. The concentration of the protein solution was varied from 0.5 mg mL1 to 10 mg mL1. Heat treated BLG was prepared at 0.1 mg mL1 in concentration and was heated in a water bath at a temperature of 80  C during 35 min. 2.2. Pulsed-light treatment PL treatments were performed using a SBS-XeMaticA-(L þ L) device (SteriBeam Systems GmbH, Kehl, Germany). For the emission of light pulses, the electric power is stored in an energy storage capacitor and later released quickly to the Xenon lamps which emit then high intensity light pulses of 325 ms duration (Lasagabaster & Martínez de Marañón, 2006). The emitted light spectrum includes wavelengths from 200 nm to 1000 nm with a considerable amount of light (approximately 40%) in the UV-C spectrum (Wekhof, 2000). Samples at room temperature (20e23  C) were placed at 8 cm from the upper Xenon lamp and received between 1 and 10 light pulses of 0.4 J cm2, up to a maximum total fluence of 4 J cm2. 10 mL of BLG solutions were poured in a quartz trough (16.6  9.8 cm) and stirred between pulses. No significant temperature increase was found at the maximum total fluence. Each experiment was repeated at least three times. 2.3. Determination of the surface hydrophobicity of glycoconjugates The surface hydrophobicity of control (native) and treated BLG was investigated by binding of 8-anilino-1-naphthalenesulfonate (ANS). The relative fluorescence intensity (FI) of the ligandeprotein conjugates was measured on a Shimadzu RF-1501 fluorescence spectrophotometer at room temperature. The wavelengths of excitation (lexc) and emission (lem) were 390 nm and 470 nm with slit widths of 10 nm. 10 mL of ANS solution (8.0 mM in 0.1 M sodium phosphate buffer, pH 7.4) was added to 1 mL of 0.1 mg mL1 and 1.5 mg mL1 of native and treated BLG samples, the resulting solution mixed and equilibrated for 2 min and, finally, the fluorescence intensity measured at room temperature. All measurements were performed in triplicate.

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2.4. Size exclusion chromatography (SEC) Size exclusion chromatography (SEC) was carried out under nondenaturing conditions (0.05 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl) using a Superdex 75 column, HR 10/30 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), on an AKTA system. A 100 mL volume of a 0.1 mg mL1 and 1.5 mg mL1 of native and treated BLG samples was applied to the column at room temperature. Elution was achieved in isocratic mode at 0.8 mL min1 for 30 min, and detection of eluting proteins was performed at 214 nm. The standard proteins used for calibration were human serum albumin (67 kDa), ovalbumin (43 kDa), alpha-chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa) (GE Healthcare Bio-Sciences AB). The void volume was determined with blue dextran 2000. 2.5. Circular dichroism (CD) Circular dichroism (CD) measurements were performed at 25  C on a Jasco J-810 spectropolarimeter using 0.2 cm or 0.01 cm path length quartz cuvettes. Protein samples were 0.1 mg mL1 or 1.5 mg mL1 in pure water. Thermal unfolding was induced by increasing temperature at a rate of 1  C min1 (using a programmable Peltier thermoelectric) and measuring the ellipticity at 205 nm or 217 nm. 2.6. NMR spectroscopy One-dimensional 1H NMR spectra were recorded at 800 MHz and 25  C on 600 mL samples containing 0.1 mg mL1 or 1.5 mg mL1 in water with 5% (by vol.) 2H2O and 17 mL TSP (sodium trimethy-silyl propionate, used as internal chemical shift reference for protons at 0.0 ppm). The protein samples were irradiated with 0 and 10 pulses previously to the addition of 2H2O and TSP. 2.7. Surface tension Surface tensions at the airewater interface of protein solutions were measured by using an FTA200 pulsating drop tensiometer (First Ten Ångstroms, USA). The capillary drop was formed with a tip of a syringe of 0.914 mm within an environmental chamber at room temperature, in which standing water increased the relative humidity to minimize drying effects. When required, changes in g (surface tension) were monitored every one second. All measurements were made at room temperature (z20  C). Surface tension was monitored at room temperature for 30 min. 2.8. Surface rheology Surface shear rheological measurements were carried out to study the mechanical and flow properties of adsorbed layers at fluid interfaces, which are sensitive to surface structure and composition (Ridout, Mackie, & Wilde, 2004). Experiments at the airewater interface were made using a stress controlled rheometer, AR2000 Advanced Rheometer (TA Instruments) and an aluminium bicone (diameter 60 mm, angle cone 4:59:13) as measuring geometry. The surface rheological response in 50 mL protein solution was tested by oscillation mode within the range of linear viscoelastic region at a frequency and strain of 0.1 Hz and 0.014, respectively. Measurements were monitored for 30 min at room temperature. 2.9. Foaming properties Foam production was achieved by using a Foamscan TM apparatus (Teclis-ITConcept, Longessaigne, France). The principle is to

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foam a 10 mL protein solution by gas sparging (N2) through a porous glass frit (flow of gas: 45 mL min1; porosity 16e40 mm). The amount of liquid incorporated in the foam and the foam homogeneity are followed by measuring the conductance in the cuvette containing the liquid and at different heights in the column by means of electrodes. Bubbling was stopped after 80 s. Drainage of the foam was followed via conductivity measurements at different heights of the foam column. The overall foaming capacity (FC) was determined by Eq. (1). Foam stability was characterized by half-life of foam t1/2 (s), the time for drainage half of the initial volume of foam (Eq. (2))

FC ¼

Vfoam ðf Þ Vgas ðf Þ

(1)

t1=2 ¼ ðk2 Vo Þ1

(2)

3. Results and discussion 3.1. Impact in structural integrity 3.1.1. Surface hydrophobicity The surface hydrophobicity of BLG was investigated by binding of 8-anilino-1-naphthalenesulfonate (ANS) to evaluate the changes of 0.1 mg mL1 and 1.5 mg mL1 treated BLG with 10 pulses (4 J cm2) and non-treated BLG. Table 1 shows that a more effective binding of the fluorescent probe was much more marked for the treated samples. Hydrophobic groups seem to be more exposed at the molecular surface as protein treatment proceeds. Hence, treated protein provoked structural changes in the protein and subsequently, it caused a more mobile structure in which hydrophobic regions initially buried in the internal structure of the native protein became more accessible to ANS binding. In principle, the more hydrophobic the proteins, the greater the decrease in interfacial tensions, which it could improve the foaming properties (Kato & Nakai, 1980). These results are not in agreement with a previous work (Elmnasser et al., 2008) in which, after a pulsedlight treatment of 22 J cm2, they found changes in the polarity of the BLG molecule from a more hydrophobic to a less hydrophobic environment. A likely reason may be that stirring between pulses was not applied in Elmnasser’s study. Stirring could help to a greater exposure of proteins to light effect and thereby to a greater modification of protein structure. Moreover, in this previous work, protein concentration (4 mg mL1) was higher than the concentration used for our study (0.5e1.5 mg mL1). Although applied fluence was higher, a “shadow effect” could arise caused by higher protein concentration. In this more concentrated scenario, upper proteins could be more easily modified and lower proteins would suffer a lower impact.

carried out to detect possible losses of secondary structure after PL treatment. The CD spectrum of non-treated BLG displays a positive extreme around 195 nm and a broad negative band centred around 218 nm, and it is essentially concentration independent (Fig. 1a and b). Such spectrum is typical of a protein with a high content of b-sheet structure, and it is consistent with secondary structure content of native BLG (which on average contains 17% a-helix, 44% b-sheet, 27% random coil and 12% turns) (Trofimova & de Jongh, 2004). Upon treatment the negative band is shifted to smaller wavelengths and the intensity at 195 nm decreases, which indicates a conformational change with a reduction in the global content of regular secondary structures and an increase in random coil conformations. This change is more pronounced at lower protein concentrations, suggesting that a high protein concentration protects from the partial denaturing induced by the light pulses. At high protein concentrations a distinct helical pattern emerges (a minimum at 208 and more negative ellipticity at 222 nm), suggesting an increase in the proportion of helical secondary structure at the expense of the b-sheet one. 3.1.3. Tertiary structure. NMR The changes in the tertiary structure can be conveniently examined in the regions of the proton spectra containing the resonances of the methyl groups whose resonances are shifted to larger frequencies (smaller ppm value) because of tertiary effects. Because of the high mobility of the methyls, at least part of them can be detected, despite of the large size of the molecule (a homodimer of 40 kDa). In the spectrum of native BLG several resolved methyls signals are observed in the range 0.7e0.0 ppm, typical of folded proteins. The intensity of these signals decreases after treatment with light pulses, while the signals at 0.9e0.8 increases and becomes broader (this is the region where most

3.1.2. Secondary structure The far-UV (ultraviolet) CD spectrum of proteins can reveal important characteristics of their secondary structure. For that reason, Far-UV circular dichroism spectra of 0.1 mg mL1 and 1.5 mg mL1 of non-treated and treated BLG (10 pulses) were Table 1 Surface hydrophobicity (S0) of native and treated (10 pulses). Sample

ANS fluorescence

Native BLG 0.1 mg mL1 Treated (10 pulses) BLG 0.1 mg mL1 Native BLG 1.5 mg mL1 Treated (10 pulses) BLG 1.5 mg mL1

15.99  0.42 23.71  0.28 11.10  0.51 23.66  2.05

Fig. 1. Far-UV circular dichroism on non-treated and treated BLG in water a) at 0.1 mg mL1 and b) at 1.5 mg mL1. The spectra were taken using cuvettes with path lengths of 2 mm (a) or 0.1 mm (b) in order to obtain a high signal to noise ratio over the same weave length range for the two samples.

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methyls are and in particular those in random coil regions of the protein). Therefore the results indicate a conformational change at the level of the tertiary structure, which is less severe at high protein concentrations (Fig. 2b). 3.1.4. Quaternary structure The quaternary structure of native and treated BLG (10 pulses) was studied at two different concentrations (0.1 mg mL1 and 1.5 mg mL1) by using size exclusion chromatography (SEC). PL treatment induced a modification of the association state of BLG molecules (Fig. 3a and b). High molecular weight (HMW) species were detected in the treated BLG, more abundant at 0.1 mg mL1 sample than in 1.5 mg mL1 sample. This result is consistent with the broadening observed in the methyl NMR signals of BLG in the region 0.9e0.8 (Fig. 2). The presence of aggregations after treatment can be due to covalent or non-covalent interactions. Nevertheless, the aggregations found in BLG by heating involve thiol-disulphide exchange reactions, leading to the formation of linear disulphide-linked aggregates of BLG monomers (Hoffmann & van Mil, 1999). Although no temperature increase occurred during the PL treatment, protein conformation showed similar changes than in a heat denaturation process, where the first step involves the dissociation of the natural dimer; it is followed by conformational changes in the monomer, leading to an increased reactivity of

Fig. 3. Size exclusion chromatography on non-treated and treated BLG a) at 0.1 mg mL1 and b) at 1.5 mg mL1.

the free thiol group. Therefore, it might be expected similar type of aggregations where subsequent aggregation of unfolded protein monomers occurs via hydrophobic interactions and thioldisulphide interchange reactions. In this case, Elmnasser et al. (2008) found similar behaviour. After a pulsed-light treatment of BLG with 10 J cm2 protein aggregation was observed, demonstrating that the aggregation was due to the formation of disulfide bonds. 3.2. Surface properties To study the impact of pulsed-light processing on the adsorption of BLG at the airewater interface caused by previously explained conformational changes, both the adsorption process itself and the surface rheological properties of the formed layer might be affected.

Fig. 2. 1H NMR spectra of non-treated (thin line) and treated (thick line) BLG in water a) at 0.1 mg mL1 and b) at 1.5 mg mL1. Only the region where most of the methyl signals appear are shown. Al the spectra were recorded with 1024 scans and are plotted with the same vertical scale for each pair with the same protein concentration.

3.2.1. The adsorption process The development of surface tension in time upon creation of an essentially protein airewater interface was studied by using the pendant drop technique. The surface tension of 0.1 mg mL1 solutions of BLG are shown in Fig. 4a. It is significant that all pulsetreated samples showed lower values than heat treated BLG which is fully denaturated. All treated samples lowered the surface tension more rapidly than non-treated samples. 10 pulse-treated

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for adsorption to the interface. It seems also that surface hydrophobicity of the molecule is greater in treated samples (Table 1). This would also affect the rate of exchange between the bulk phase and the interface, leading to an increase in residence time at the surface and thus a faster formation of an interfacial layer (Martin, Grolle, Boss, Stuart, & van Vliet, 2002). It is clear that the degree of unfolding, related to an increase of protein flexibility and loss of tertiary structure together with the resulting increase in surface hydrophobicity has a major influence in surface activity. 3.2.2. Surface rheology The impact of the pulsed-light processing in the surface rheological properties of BLG was then investigated. Fig. 5a shows the shear elastic modulus of 0.1 mg mL1 solutions of BLG measured over a period of 30 min. It can be observed that G0 increased rapidly at short times followed by slow increase at longer times for all the treatments. The increase in surface rheology with time is considered to be a result of protein adsorption at the interface, conformational changes at the interface and intermolecular interactions (Martin, Bos, & van Vliet, 2002). However, BLG solutions, fully denaturated by thermal treatment, showed less elastic interfaces than 4 and 10 pulse-treatments. The effect of PL in the surface shear elasticity shows than treated samples (4 pulses and 10 pulses) forms a stronger interface than the non-treated sample. This stronger interface formed with the samples treated with 4 and 10 pulses is repeated at higher concentrations up to 10 mg mL1

Fig. 4. The surface tension of 0.1 mg mL1 BLG versus time (a) and effect of concentration on the surface tension (taken at 30 min) as measured by the pendant drop technique for BLG with different pulsed-light treatments and heat treatment (b); () BLG non-treated; (6) BLG treated 1 pulse; (C) BLG treated 4 pulses; (-) BLG treated 10 pulses.

and 4 pulse-treated solutions lowered the surface tension more rapidly than 1 pulse-treated and non-treated solution. The rate of adsorption in 10 pulse-treated and 4 pulse-treated solutions then became similar. After 30 min adsorption the surface tension of both treated solutions was around 51 mN m1, 3 mN m1 and 6 mN m1 lower than 1 pulse-treated and non-treated solution. This trend of lower surface tension values with 4 and 10 pulses solutions was maintained throughout a range concentration from 0.1 mN m1 to 10 mN m1, although at highest concentrations, differences are less noticeable (Fig. 4b). The surface activity was definitely higher in treated samples. The ability of water-soluble proteins to reduce surface tension is defined by many factors such as initial protein concentration in the bulk, protein diffusion coefficient that depends on the hydrodynamic size (Croguennec et al., 2006) or net charge on the molecule which discourages the formation of a compact adsorbed layer due to the charge repulsion (Wilde, 2000). Another important factor is the flexibility of the adsorbing protein molecule, which it seems it is influenced by the amount of light dose. Secondary structure and tertiary structure clearly showed a partial denaturation in treated samples which gave a more flexible structure. The more flexible the molecule is, the more rapidly it can adopt a conformation suitable

Fig. 5. Shear elastic modulus of 0.1 mg mL1 BLG versus time (a) and effect of concentration on the surface rheological properties (taken at 30 min) as measured by surface shear rheology for BLG with different pulsed-light treatments and heat treatment (b); () BLG non-treated; (6) BLG treated 1 pulse; (C) BLG treated 4 pulses; (-) BLG treated 10 pulses.

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(Fig. 5b). The data support the idea that the enhanced modulus observed in the adsorption experiments for BLG is owing to the presence of greater surface concentrations of protein at a given adsorption time. As more protein adsorbs, the probability of entanglement between neighbouring molecules is increased (Cornec, Kim, & Narsimhan, 2001) and then elastic modulus is higher, as it happens in the treated samples. PL treatment induced drastic structural changes in the secondary and tertiary structures. Those conformational changes and the increase in surface hydrophobicity and molecular flexibility are closely related to the increase in the viscoelasticity of the interface. The rapid increase of the shear elastic constant indicates faster and probably different intermolecular associations between proteins. 3.3. Foaming properties Foaming properties of native and PL treated proteins at different concentrations were finally studied after pulsed-light processing (Figs. 6 and 7). At low concentrations of BLG (0.5 mg mL1 and 1.5 mg mL1), differences in foaming capacity (Fig. 6) were more significant amongst treated samples at different levels, whereas at high concentrations (5 mg mL1 and 10 mg mL1) showed more similar values. For example, at 0.5 mg mL1, after a pulsed-light treatment of BLG with 1 and 4 pulses, foaming capacity of BLG increased approximately 18% from the values of non-treated BLG. Furthermore, at the same concentration, after 10 pulse treatment, foam capacity increases 34% comparing to non-treated BLG. As sample concentration increased, differences of foam capacity between treated and non-treated samples decreased, reaching a maximum concentration (10 mg mL1) where, comparing to native protein, treated samples with 1 pulse showed slightly lower values and similar values with 4 pulses, although treated samples with 10 pulses maintained a considerable difference regarding nontreated protein (z12%). Static foam stability was followed by the half-life time, which is the volume of liquid drained from the foam at half of the total time and it corresponds to the empirical second-order equation (Eq. (1)). Fig. 7 shows the foam stability as a function of BLG concentration. Overall, the foam stability increased with protein concentration for all the samples (treated and non-treated) but with significant differences among them. It seems that the samples treated with 1 pulse did not really change comparing to the native protein. However, it was rather evident that, from a treatment with 4 pulses, BLG foaming capacity considerably increased and showing the

Fig. 7. The effect of pulsed-light processing at different levels of light dose on foaming stability of BLG at different concentrations; () BLG non-treated; (6) BLG treated 1 pulse; (C) BLG treated 4 pulses; (-) BLG treated 10 pulses.

highest differences with a treatment of 10 pulses. At 4 and 10 pulses, foaming stability behaved differently to foaming capacity at higher concentrations. In this case, foaming stability values increased as protein concentration was higher, reaching the maximum differences at 5 mg mL1, where foaming stability of the sample treated with 10 pulses increased more than twofold comparing to the non-treated sample. Foaming properties seems strongly correlated to the rate of protein adsorption to the airewater interface (Figs. 4 and 5) and thereby to the decrease of surface tension, giving a higher foaming capacity. Treated proteins show the ability to adsorb more rapid to the interface, mainly caused by an increase in the surface hydrophobicity and a higher flexibility due to partial denaturation. The high initial viscoelastic properties (Fig. 5) of the interfacial film of treated proteins resist to the shear stress resulting from liquid drainage. Additionally, BLG aggregates (Fig. 3) may improve the foamability and foam stability under certain conditions. More precisely, the improvement of the foaming properties was found to depend on the size and on the ratio between protein aggregates and non-aggregated proteins (Rullier, Axelos, Monique, Langevin, & Novales, 2009). When aggregates adsorb at the interface, they can cross-link the two thin films of adjacent bubbles, leading to stable films. Such oligomeric proteins could form an ordered structure, which would change the foaming properties of the samples (Wierenga, van Norél, & Basheva, 2009). These results clearly reveal, contrary to other works (Elmnasser et al., 2008), significant conformational changes in this protein due to the PL treatment and consequently, significant changes take place in the functionality of treated BLG. 4. Conclusions

Fig. 6. The effect of pulsed-light processing at different levels of light dose on foaming capacity of BLG at different concentrations; () BLG non-treated; (6) BLG treated 1 pulse; (C) BLG treated 4 pulses; (-) BLG treated 10 pulses.

Results showed that treated samples were affected in their structure by PL treatment changing their conformation which it is likely to increase in random coil. This loss of secondary and tertiary structure suggests that partial denaturation took place after PL treatment. Furthermore, hydrophobic groups seemed to be more exposed at the molecular surface as protein treatment proceeds which leaded the presence of high molecular weight species. All these conformational changes were responsible of having higher rates of protein adsorption at the airewater interface, and thus, treated samples showed higher surface activity. Treated protein also showed a significant increase in the viscoelasticity of the adsorbed layer. Higher foaming capacity in treated protein seemed

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strongly correlated to the rate of protein adsorption at the aire water interface. Similarly, the enhanced viscoelastic properties of the adsorbed layer slowed liquid drainage which resulted in higher foaming stability. Results obtained in the present work show that PL technology is potentially an efficient non-thermal process not only in inactivating a wide broad of microorganisms but for improving the foaming properties of solutions with high protein content such as whey. Therefore, the implementation of this technology in some food industry sectors could be worthwhile for the production of high added-value ingredients. Further work is already focused on the design of a pulsed-light dynamic device for liquid decontamination to assess the real potential of this technology in the food industry to apply it in full-scale production. The study of the effect of PL on other functional properties like emulsifying and gelling properties is also needed. Acknowledgements This work was financial supported by the Department of Industry, Trade and Tourism from the Basque Government and by the Ministry of Industry, Tourism and Trade from the Spanish Government. M. L. Artíguez was funded by a PhD grant of the Department of Education, Universities and Research from the Basque Government. References Anema, S. G., & Li, Y. M. (2003). Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size. Journal of Dairy Research, 70, 73e83. Barbosa-Cánovas, G. V., Góngora-Nieto, M. M., & Swanson, B. G. (1998). Non thermal electrical methods in food preservation. Food Science and Technology International, 4, 363e370. Cornec, M., Kim, D. A., & Narsimhan, G. (2001). Adsorption dynamics and interfacial properties of alpha-lactalbumin in native and molten globule state conformation at airewater interface. Food Hydrocolloids, 15(3), 303e313. Croguennec, T., Renault, A., Bouhallab, S., & Pezennec, S. (2006). Interfacial and foaming properties of sulfydryl-modified bovine b-lactoglobulin. Journal of Colloid and Interface Science, 302(1), 32e39. Davis, J. P., Doucet, D., & Foegeding, E. A. (2005). Foaming and interfacial properties of hydrolyzed b-lactoglobulin. Journal of Colloid and Interface Science, 288(2), 412e422. Dickinson, E., & Wasan, D. T. (1997). Food colloids, emulsions, gels and foams. Current Opinion in Colloid & Interface Science, 2(6), 565e566.

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