Interfacial and emulsifying behaviour of crayfish protein isolate

LWT - Food Science and Technology 44 (2011) 1603e1610

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Interfacial and emulsifying behaviour of crayfish protein isolate Alberto Romero a, *, Valérie Beaumal b, Elisabeth David-Briand b, Felipe Cordobés a, Marc Anton b, Antonio Guerrero a a b

Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 Sevilla, Spain UR1268 Biopolymères Interactions Assemblages, Equipe Interfaces et Systèmes Dispersés, INRA, F-443316 Nantes, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 June 2010 Received in revised form 19 February 2011 Accepted 7 March 2011

The interfacial behaviour of adsorbed protein films constituted with a crayfish protein derivate that is typically produced as by-product from the food industry, has been studied at the airewater and oilewater interfaces. An analysis of the surface pressure under compression-expansion cycles of this protein was carried out as a function of time, concentration and pH (2 and 8). Besides, interfacial tension and adsorption kinetics also were determined as a function of time at different concentrations and pH values. Interfacial rheological properties were studied under dilatational deformations applied to a single droplet, either at the initial step of film formation or once the interfacial tension was at equilibrium and the film was completely formed. The contribution of the interfacial properties to the behaviour of oil-in-water emulsions stabilised with this protein derivative were also analysed. Finally, droplet size distributions obtained for concentrated emulsions stabilised by crayfish protein were analysed and related to the interfacial tension behaviour. We have demonstrated that crayfish proteins at pH 8 show higher solubility, smaller aggregates and better interfacial activity (higher surface pressure and lower interfacial tension) with higher interfacial viscoelasticity, than at pH 2. A two-dimensional model of the results showed that oilewater and airewater interfaces are clearly related to the improved stability of emulsion made with crayfish proteins at pH 8. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Emulsion Crayfish protein Interfacial tension Surface pressure Interfacial rheology

1. Introduction Much of the food industries, particularly those involved in primary production, generate large amounts of waste and byproducts. Some of the by-products are being evaluated with the aim of producing added value products (i.e. biomaterials, functional foods or other food applications). One major application for these refined by-products, particularly plant proteins, is in the production of emulsions (Bengoechea, Cordobés, & Guerrero, 2006; Chen, Dickinson, Langton, & Hermansson, 2000; Ralet & Guegen, 2000;) or fish (Cofrades, Carballo, Coreche, & Colmenero, 1996; Romero, Cordobés, Puppo, Guerrero, & Bengoechea, 2008). A primary requirement for a wide variety of industrial applications of emulsions is stability, which is highly influenced by the role of protein in the two-phase system. Thus, oil/water emulsions are complex systems whose properties depend on the specific characteristics of the three component parts: the oily disperse phase, the continuous aqueous

* Corresponding author. Tel.: þ34 954557179; fax: þ34 954556447. E-mail address: [email protected] (A. Romero). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.03.005

phase and the interface. Thus, it is necessary to consider the properties of each phase, but it is essential to focus specifically on the interface to understand the properties of the emulsion. The interface of an oil/water emulsion for food applications is typically composed of proteins (in a broad range of molecular weights), or low molecular weight emulsifiers (monoglycerides, esters, phospholipids, etc.) or a mixture of both (Dalgleish, 2006). Proteins, because of their amphiphilic nature tend to reduce the interfacial tension (McClements, 2004; 2005). For this reason proteins can be used as functional ingredients for the formation and stabilization of food emulsions and foams (Kinsella, 1976; Norde, 2003). During the emulsification process, proteins are rapidly adsorbed on the surface of the droplets, forming an interfacial layer that protects droplets against destabilization mechanisms such as coalescence (Bos & Van Vliet, 2001; Dickinson, 1999; Wilde, Mackie, Husband, Gunning & Morris, 2004). Studies on emulsion behaviour can be related to the physical properties of the two-dimensional model interfaces: oilewater but also airewater (Ficher & Enri, 2007). Important aspects regarding potential interfacial surface active molecules are: their capacity to lower the interfacial tension and the rate of lowering; the equilibrium adsorbed amount, their ability to desorb, the possibility to change

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their conformation during and after adsorption, the thickness of the adsorbed layer, the interaction between the adsorbed molecules, and their lateral mobility. However, most important for emulsions and foams is the actual value of the static/dynamic interfacial or surface tension as a function of the interfacial deformation corresponding to the response of the film against a deformation (Bos & Van Vliet, 2001). In fact, there are some studies exploring the relation between viscoelasticity of surfactant adsorption layers and stability of emulsions and foams (Das & Kinsella, 1990; Dickinson & Hong, 1994; Dickinson, Owusu, Tan & William, 1993; Edwards, Brenner, & Wasan, 1991). However, actual data linking the former to the latter are scarce. The main problem is that all measurements of interfacial rheology are made on macroscopic surfaces, and the range of stresses, strains and rates of strain applied certainly do not reflect the turbulent nonequilibrium conditions of practical foam formation or emulsification. Fortunately, a number of methods have become available by which interfacial rheological behaviour can be studied under nonequilibrium conditions at intermediate strain rates both for oilewater and airewater interfaces (Benjamins & van Voorst Vader, 1992; Benjamis Cagna & Lucassen-Reynders, 1996; BerginkMartens, Bos, & Prins, 1993; Joos & Van Uffelen, 1995; Prins, 1995; Van Aken & Merks, 1996). Two developed techniques (the barrierand-plate and the dynamic drop tensiometer methods) are mainly used to determine the interfacial viscoelastic modulii. Although both methods are quite different in the experimental setup, the results are likely to be similar (Benjamins, Cagna, & LucassenReynders, 1996). Possibly, these and other yet more advanced methods may give in the future, a better insight into the relationship between emulsion and foam behaviour and interfacial rheology. The overall objective of this study is to evaluate crayfish protein potentials as emulsifier in relation to its air/water and oil/water interfacial behaviour, which may help to select optimal conditions in order to enhance emulsion stability. More specific objectives are to establish a relationship between parameters from oilewater and airewater interfaces, as well as between O/W interfacial tension and viscoelasticity properties, at different pH values and protein concentration. Eventually, a further objective is to establish a link between those interfacial results and crayfish-based emulsion stability. 2. Material and methods 2.1. Materials Crayfish flour (CF) was manufactured at pilot-plant scale by ALFOCAN S.A. (Isla Mayor, Seville, Spain). Crayfish meat was separated from the exoskeleton and comminuted to form a meat slurry that was dried at 150e160  C in a rotary drum dryer, to obtain a low moisture crayfish powder. The flour supplied by ALFOCAN S.A. consisted of 64 wt% protein, 19 wt% lipids, 13 wt% ashes and 4 wt% moisture. All chemicals used were of analytical grade purchased from Sigma Chemical Company (St. Louis, MO, USA). Distilled water was used for the preparation of all solutions. 2.2. Methods 2.2.1. Preparation of crayfish protein isolate Crayfish protein isolate (CFPI) was prepared from the crayfish flour by solvent extraction of lipids, alkaline extraction of soluble proteins and isoelectric precipitation, according to the procedure described in a previous paper (Romero, Cordobés, Puppo, et al., 2009). The crayfish flour powder was sieved, defatted by hexane, air-dried at room temperature and stored at 4  C until used. The defatted flour was dispersed in water (10% w/v) giving rise to a pH

of 6.6, that was adjusted to pH 10.5 with 25% NaOH. The dispersion was stirred at room temperature for 4 h and centrifuged at 900 g for 25 min at 4  C in an RC5C Sorvall centrifuge (Sorvall Instruments, Wilmington, DE, USA). The supernatant was then adjusted with 6 N HCl to pH 3.4, which corresponds to the actual pI of crayfish protein as determined from zeta potential measurements (Romero, 2009), and centrifuged at 9000 g for 10 min at 4  C. The pellet was washed and resuspended with distilled water. The protein dispersion was freeze-dried in a Freeze Mivile 3 (VIRTIS, USA). 2.2.2. Chemical composition of crayfish protein isolate The protein content was determined in quadruplicate as %N  6.25 using a LECO CHNS-932 nitrogen micro analyzer (Leco Corporation, St. Joseph, MI, USA) (Etheridge, Pesti, & Foster, 1998). Lipid content was analysed in triplicate by Soxhlet extraction. Moisture and ash content of the isolate was determined in quadruplicate by AOAC, 1995 approved methods. CFPI, obtained according to the above described isolation procedure, consisted of 90.61  2.14 wt% protein, 0.96  0.08 wt% lipids, 3.98  0.24 wt% ash and 4.45  wt% moisture. 2.2.3. Determination of protein isoelectric point (pI) and solubility For the determination of the pI of the aqueous crayfish flour dispersions (0.96 g protein/40 mL) were prepared and the pH of different aliquots was adjusted with 6 N NaOH or 2 and 6 N HCl to produce either alkaline or acid pHs. These solutions were equilibrated for 1 h at room temperature under continuous stirring in an IKA Magnetic Stirrer, RCT model. Four millilitres of each solution was taken for the determination of the initial protein content. The remaining solution was centrifuged for 20 min at 10,000 g, at 10  C. The supernatants were collected for protein content determination by means of the above mentioned LECO nitrogen micro analyser method. Percentages of soluble protein (calculated as protein content of supernatant  100/weight of CFPI powder) were plotted vs. pH to determine the pI. 2.2.4. Atomic Force Microscopy (AFM) A drop of 1 g/L of CFPI solution was deposited on a mica plate by means of a Pasteur pipette for each pH value, with two replicates for each pH value. A compressed nitrogen stream was used in order to dry the sample, achieving a homogeneous distribution on the mica plate. After that, the mica plate was kept in a desiccator overnight. AFM imaging of samples was performed by using an AFM CP (Park Scientific Instruments, USA) under the tapping mode using a cantilever. 2.2.5. Determination of airewater interface characteristics Measurements of the surface pressure (P)-surface area (A) isotherms were determined by compressioneexpansion cycles by the Wilhelmy plate method on an isolated and fully automated Langmuir-type film balance (KSV 300 V2, Helsinki, Finland). The balance consisted of two movable barriers that surrounded the Wilhelmy plate. The area of the vessel, initially of 350 cm2, was reduced to 40 cm2 at the end of compression. The vessel was filled with each of the buffer systems used for emulsion preparation. Temperature was kept at 20  C by circulating water from a Bioblock Ministat thermal bath (Illkirch, France). The cleanliness of the buffers was checked by a compression and expansion cycle using the buffer without protein injection. The surface was deemed to be clean when the surface pressure rise was negligible (less than 0.05 mN/m). The barrier speed was fixed at 40 cm2/min. The deposition of the drops was performed with a micrometric syringe to avoid thick smears and located on the surface. To allow the processes of spreading, adsorption and rearrangements of proteins,

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samples were left to stand for 40 min before compression. The compression speed was maintained constant at 28.4 cm2/min, which is sufficiently low to prevent secondary effects due to the barrier displacement. These measurements were performed at different protein concentrations and pH values (2 and 8). At least three isotherms were performed for each sample. 2.2.6. Determination of oilewater interface characteristics Transient interfacial tension and surface dilatational parameters were carried out using a drop tensiometer from IT Concept (Longessaigne, France). An axisymmetric drop (purified n-hexadecane) was formed at the tip of the needle of a syringe whose verticality was controlled by a computer. The drop profile was digitized and analyzed through a CCD camera coupled to a video image profile digitizer board connected to a computer. The image was continuously visualized on a video monitor. Drop profiles were processed according to the Laplace equation as was described by Castellani, Al-Assaf, Axelos, Phillips, and Anton (2009). All the experiments were carried out, at least in duplicate, in an optical glass cuvette (8 ml), containing the corresponding aqueous solution of protein. The system was thermostated at 20  0.1  C. Surface tension kinetics and sinusoidal area fluctuation experiments were carried out independently. Surface tension kinetics were performed for different protein concentrations, from 0.1 to 1.0 g/L. Whilst, the viscoelastic modulii of the protein adsorption layers were determined at non-equilibrium conditions at 0.126 rad/s and under equilibrium at different frequencies (0.031, 0.062, 0.126 and 0.314 rad/s). The protein concentration was fixed at 1.0 g/L at which the O/W interface was saturated. 2.2.7. Emulsion processing Different 50% (p/v) sunflower oil emulsions were prepared using 3.00 wt% CFPI and water. First of all, CFPI powder was solved in the corresponding buffer. Then, oil was added to the aqueous protein solution and the two-phase system was homogenised for 1 min at 20,000 rpm. using a polytron PT 3000 homogeniser (Kinematica, Switzerland) equipped with a 12 mm dia. head. Homogenisation of the emulsion premix was then achieved with a two-stage highpressure valve homogeniser (TC5, Stansted Fluid Power, UK) at 15 MPa for 5 min. The emulsion (40 ml) was left recirculating in the homogeniser for 3 min at a flow rate of 120 ml/min. 2.2.8. DSD measurements Measurements of droplet size distribution (DSD) were performed in a Saturn Digisizer 5200 from Micromeritics (USA). For this purpose, 0.5 ml of emulsion was taken and diluted in 11.5 mL of 0.05 M, pH 8 TriseHCl buffer with 1% SDS in order to facilitate disruption of the flocs prior to any DSD measurement (Puppo et al., 2005). Values of the Volumetric mean diameter, d4,3, which is inversely proportional to the specific surface area of droplets, were obtained as follows:

P n dx dx;y ¼ P i yi ni di

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2.2.9. Interfacial protein concentration Non-adsorbed proteins were washed from the oil droplets following a method adapted from the procedure described by Patton and Huston (1986). Each fresh emulsion (2 ml) was diluted in 2 ml sucrose solution (500 mg/ml, with the same pH value as the aqueous phase of the emulsion: pH 2.0 and 8.0). This dilution (2 ml) was then carefully deposited at the bottom of a centrifuge tube containing 10 ml of a buffer solution with the same pH and NaCl concentration as the respective emulsion. The tubes were then centrifuged at 3000 g for 2 h at 10  C. After centrifugation, three phases were observed in the tubes: the creamed oil droplets at the top, the intermediate buffer solution, and the aqueous phase of the emulsion deprived of oil droplets at the bottom. The tubes were frozen (20  C) and cut so as to recover the three phases. Proteins from the upper phase were the adsorbed proteins and those from the bottom phase are unadsorbed proteins. When the middle phase was turbid due to the presence of small oil droplets, the protein stabilizing the droplets was aggregated to adsorbed proteins from the upper layer. Protein concentration was assessed in the bottom of the tubes by the procedure of Markwell, Hass, Bieber, and Tolbert (1978). Protein concentration was referred to emulsion volume, and interfacial protein concentration (G, mg/m2) was calculated as follows:

G ¼

Cap SV

(3)

where Cap is the protein concentration (mg/ml emulsion) and SV is the specific interfacial area (m2/ml emulsion). This procedure was determined by Martinet, Saulnier, Beaumal, Couthaudon, and Anton (2003). 2.2.10. Statistical analysis Three replicates of each measurement were carried out. Statistical analyses were performed using t-test and one-way analysis of variance (ANOVA, p < 0.05) by means of the statistical package SPSS 18. Standard deviations from some selected parameters were calculated.

3. Results and discussion 3.1. Protein solubility Fig. 1 shows the typical U-shaped solubility curve for the CF protein isolate as a function of pH. AFM images for 1% wt protein

(1)

where ni is the number of droplets with a diameter di. The uniformity ratio is an index of polidispersity of the different droplet sizes, defined by the following expression:

P U ¼

Vi jdðv; 0; 5Þ  di j P dðv; 0; 5Þ Vi

(2)

where d(v,0,5) is the median for the distribution, and Vi is the volume of droplets with a diameter di.

Fig. 1. Solubility profile (Solubility % vs. pH) of crayfish flour protein isolate with AFM images for 1% wt of protein solution at pH 2 and 8.

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Fig. 2. Interfacial pressure at the A/W interface by a compressioneexpansion cycle as a function of the reciprocal of surface concentration A) pH 2 and B) pH 8 (,: 10 mg/mL; B: 15 mg/mL; : 20 mg/mL; 7: 25 mg/mL; : 30 mg/mL).

dispersions at the two studied pH values also can be observed in this figure. As it is well known, the minimum in protein solubility observed in Fig. 1 at pH ca. 3.4 corresponds to the isoelectric point of this protein isolate. This value is almost coincides with the pI estimation that was previously determined by particle charge measurements as a function of pH (Romero, 2009). Electrostatic

interactions contribute to increased solubility at pHs that are higher or lower than the pI, particularly at alkaline pH values. AFM images show a significant amount of relatively large aggregates at both pH values (pH 2 and 8) that can be attributed to the high degree of protein denaturation achieved during pilot plant-scale protein extraction (Romero et al., 2008). Besides, it can be observed that

Fig. 3. Interfacial tension kinetics of CFPI solutions as a function of protein concentration at different pH values: A) and B) pH 2 (e 0.25 g/L; e 0.5 g/L; ∙∙∙∙∙ 0.75 g/L; ∙∙ 1.0 g/L; ,: 0.25 g/L; 0.50 g/L; : 0.75 g/L; : 1.00 g/L) and C) and D) pH 8 (e 0.10 g/L; —— 0.25 g/L; ∙∙∙∙∙ 0.50 g/L; ∙∙ 0.75 g/L; ∙∙∙∙1.00 g/L; ,: 0.10 g/L; 0.25 g/L; : 0.50 g/L; : 0.75 g/L; : 1.00 g/L).

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aggregates are more spherical and larger at pH 2. This fact can be related to the lower solubility and the proximity of pH 2 to the isoelectric point.

3.2. Interfacial analysis 3.2.1. Airewater interface Fig. 2 shows the P-A isotherms obtained at 20  C by double compressioneexpansion cycles using the Langmuir trough method. These experiments, which allow the analysis of the behaviour of crayfish proteins at a planar airewater surface, were carried out at different pH and protein concentrations. The crayfish monolayers exhibited a liquid-expanded-like structure under the experimental conditions where it was possible to observe the monolayer collapse at the highest surface pressure. Besides, isotherms were displaced towards lower values of the reciprocal of surface concentration as the amount of protein was raised, and followed an asymptotic evolution for the two studied pH values. This evolution indicates that protein aggregation is taking place with increasing concentration, which is more remarkable at pH 2, at which larger aggregates can be observed in Fig. 1. Moreover, no further evolution in interfacial pressure can be observed above 30 mg protein which indicates that the air/water interface seemed to be saturated. At this protein concentration, the protein film showed a collapse pressure of about 30 and 35 mN/m at pH 2 and 8, respectively. These values are similar to those found by Rodriguez Patino, Sánchez, & Rodriguez Niño (1999) for b-casein. The higher surface coverage found for crayfish films when the aqueous subphase was acidic must be related to the lower level of repulsive interactions between protein residues. As, pH 2 is closer to the isoelectric point than pH 8, the results suggest that electrostatic repulsions must be more important at alkaline conditions. 3.2.2. Oilewater interface Fig. 3 shows the transient interfacial tension obtained for crayfish protein solutions as a function of protein concentration at pH 2 and 8. In both cases, a typical decrease in interfacial tension took place with time (Fig. 3A and B). Although, the interfacial tension of the system seems to be still evolving after protein adsorption for

Fig. 4. Interfacial tension at the O/W interface at the equilibrium for CFPI at different pH values as a function of protein concentration (, pH 2; - pH8).

5 h, the reduction in interfacial tension was extremely slow after the first 2 h (Fig. 3A and B). As a consequence, the value obtained after 5 h may be taken as the tension achieved at the equilibrium state, seq. These results are in agreement with those of Beverung, Radge and Blanch (1999), obtained for different proteins (ovalbumin, b-casein, lysozyme, BSA, different lipases and glutamate dehydrogenase). In all these cases the limiting value of interfacial tension was reached at the same order of magnitude in time. The same adsorption kinetics are plotted on a semi-logarithmic scale, as shown in Fig. 3C and D, in order to obtain an improved definition of the initial adsorption process. Adsorption kinetics may be generally divided into 3 stages (Beverung, Radke, & Blanch, 1999), where the first stage corresponds to an induction period. As may be observed in Fig. 3, the beginning of the crayfish protein adsorption was so fast, that the first stage could not be observed (either using linear or semi-logarithmic plot). This effect was also observed by Beverung et al. (1999) for the highest protein concentration studied. The second and third stages, which

Fig. 5. Dilatational modulii for 1 g/L of CFPI and a different pH values: A) Evolution with time at 0.12 rad/s; B) Evolution with frequency at the equilibrium (- E0 pH2; , E0 pH2; C E0 pH8; B E0 pH8).

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Table 1 sat Interfacial A/W and O/W parameters (Psat surface pressure at saturation, seq * complex dilatational modulus at equilibrium, equilibrium tension at saturation, Eeq tan deq, loss tangent at equilibrium) for 1 wt% CFPI at pH 2 and 8. pH

Psat (mN/m)

sat seq (mN/m)

* (mN/m) Eeq

tan deq

2 8

31 35

9.3 8.8

14.7 16.4

0.215 0.134

respectively correspond to a steep and moderate reduction in interfacial tension can be clearly distinguished for the two pH values. The comparison of interfacial tension at equilibrium (seq) as a function of protein concentration can be observed in Fig. 4. A decrease in seq may be observed as protein concentration was increased, showing a tendency to a plateau value, independent of the protein content, which may be denoted as the equilibrium sat ). This evolution turned out to be very tension at saturation (seq similar for the two pH values studied. In fact, saturation of the oilewater interface was reached at the same concentration (ca. 1 mg/mL). However, the decrease that took place at pH 8 was faster and more remarkable (Fig. 3), leading to lower values of interfacial sat sat ¼ 9.3 mN/m at pH 2 and seq ¼ 8.8 mN/m at pH 8) tension (seq (Fig. 4). These results would suggest that the adsorption was favoured at alkaline pHs. As is well known, the reduction in interfacial tension by the protein molecules significantly favours the emulsification process, but once the protein films are formed at the interface, viscoelastic properties of these interfacial films play a remarkable role in the stabilization of the oil droplets formed in the process (Yampolskaya & Platikanov, 2006). The viscoelastic behaviour of interfacial films formed by crayfish protein samples was evaluated throughout the adsorption process. Fig. 5 shows the values obtained for the dilatational modulii after addition of 1 mg/mL crayfish protein and subjecting an oil drop to dilatational dynamics measurements at constant strain amplitude. As may be noticed in Fig. 5A, crayfish protein films exhibited a clear elastic response with the elastic

Fig. 6. Dilatational modulus (logarithmic scale) versus interfacial tension values (linear scale) of aqueous solutions at 1.0 g/L (pH 2 and 8) at oilewater interface (, pH 2; pH8). All results were fitted by a linear regression.

modulus E0 completely dominating the storage modulus E00 . In fact, the phase angle remained below 15 throughout the whole experiment, and showed an apparent tendency to decrease. Once again, only two of the three different regions generally described for adsorption kinetics at the oilewater interface may be distinguished from the evolution of the dilatational elastic modulus. Thus, the aforementioned induction stage was not observed and a rapid increase in E0 , corresponding to the second region, took place from the beginning of the experiments. It is worth mentioning that the second region deduced from dilatational measurements coincided reasonably well with the same region detected from the interfacial tension reduction kinetics (Fig. 3). Finally, at the third stage, there was only a slight evolution of the

Fig. 7. Evolution of droplet size distribution for emulsions containing 50 wt% oil and 3 wt% CFPI as a function of storage time at (A) pH 2 and (B) pH 8 (- 1 day; , 20 days).

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elastic modulus, showing a tendency to an equilibrium value for each of the two pH values studied. The increase in elastic modulus with time may be related to the development of inter-protein contacts between neighbouring adsorbed molecules. In fact, crayfish protein showed a lower modulus in comparison with other globular proteins (data not shown). Cascao et al. (2003) reported similar low storage modulus values for b-casein, attributing this behaviour to the flexibility of b-casein proteins compared to that of globular proteins such as BSA. This flexibility would favour compression at the interface. Myofibrillar proteins are also more flexible than globular proteins, which may explain the low storage modulus values obtained. Fig. 5B shows the dilatational modulii as a function of the oscillatory dilatational frequency, performed at the end of the third stage, when the adsorption process is essentially finished. The viscoelastic moduliiefrequency curves obtained at both pH values were very similar, showing a solid-like behaviour, with a clearly predominant elastic response and a low frequency-dependence for E0 . As may be observed in Fig. 5A and B, the elastic modulus is generally higher at pH 8, which also corresponds to lower interfacial tension values and smaller protein aggregates. 3.2.3. Relationship between interfacial analysis Table 1 shows some parameters corresponding to airewater and oilewater studies. As can be deduced from this table, results from the P-A isotherms and from pendant drop interfacial tension measurements are correlated, indicating that the behaviour of crayfish proteins are similar at the airewater or oilewater interface. This coincidence has been observed also for other protein systems (Castellani, Belhomme, David-Briand, Guérin-Dubiard, & Anton, 2006). In the case of the oilewater interface studies, two aspects were considered: interfacial tension, related to the amphiphilic character and flexibility of molecules, and interfacial film rheology, related to the capacity of interactions between adsorbed molecules and the structural stability of the proteins (Cascao et al., 2003). As described by Benjamins et al. (1996) these two aspects could be connected for specific interfacial active systems. Thus, these authors reported a relationship between interfacial tension and the dilatational modulus (on a log-scale) for several proteins (ovalbumin, bovin serum albumin and sodium caseinate). Furthermore, these authors related the different slope obtained for each protein to the structural characteristics of each protein film. Fig. 6 shows how this relationship is also followed by crayfish protein interfacial films, showing the same slope at both pH values. These results suggest that pH did not bring about marked differences in the structural characteristics of the crayfish protein films. 3.3. Emulsions: DTG and interfacial concentration Fig. 7 shows the DSD profiles obtained for 50% w/v emulsions stabilised by 3 wt% CFPI, as a function of ageing time, at pH 2 (Fig. 7A) and 8 (Fig. 7B). An evolution of these profiles towards larger droplets with a tendency to the development of a second mode generally takes place, as a consequence of coalescence phenomena. Once again, the best results were found for the emulsion at pH 8, where no significant evolution with time may be noticed. Table 2 shows some parameters obtained from DSD analysis: the volumetric diameter measured at 1 and 20 days after emulsification and the uniformity ratio of fresh emulsions; as well as two parameters related to the composition of the oilewater interface: the percentage of adsorbed protein to the oilewater interface (AP) and the interfacial protein concentration (G). As may be seen in this table, the higher the electrostatic interactions obtained at pH 8 compared to pH 2 (as a consequence of the further distance from the isoelectric point), the higher the degree of

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Table 2 Parameters (d4,3 (1d.) volumetric mean diameter at one day, d4,3 (20d.) Volumetric mean diameter at twenty days, U uniformity ratio, AP adsorbed protein, G interfacial protein concentration) for emulsions containing 50 wt% oil and stabilised by 3 wt% CFPI and different pH values. pH

d4,3 (1d.) (mm)

d4,3 (20d.) (mm)

U

AP (%)

G(mg/m2)

2 8

0.33 0.30

0.43 0.31

0.68 0.54

68.6 78.4

1.5 1.6

protein adsorption at the oilewater interface (deduced from AP and G values), the lower the emulsion droplet sizes, the lower the dependence on ageing time and the lower the emulsion polydispersity (deduced from U values), all of which were consistent with the lower size of the aggregates obtained at alkaline pH. 4. Conclusions Crayfish proteins show a solubility behaviour that strongly depends on the pH value. However, protein molecules aggregate either at acidic or alkaline pH with respect to the pI, which has been previously related to protein denaturation as a consequence of heat processing (Romero et al., 2008). Near the isoelectric point these aggregates are larger, which leads to lower solubility, and show a higher sphericity, due to the lack of electrostatic interactions. A relationship between parameters from oilewater and airewater interfaces, which depends on pH values and concentration below interfacial saturation, has been found. In fact, higher values of surface pressure of the airewater interface correspond to lower values of oilewater interfacial tension. Crayfish proteins confer an elastic dominant behaviour to oilewater interfaces showing low interfacial tension values (related to the adsorption at the interface). However, these proteins show a relatively low complex dilatational modulus (related to deformation resistance) in comparison to the values found for globular proteins. Moreover, a relationship between dilatational modulus and interfacial tension, which is independent of pH, has been found. Although emulsions at pH 2 and 8 show the same interfacial protein concentration, systems at pH 8 exhibit smaller aggregates, higher solubility and better interfacial values (higher surface pressure and lower interfacial tension) with higher interfacial viscoelasticity. These results are in agreement with those shown by emulsions stabilized by the same protein concentration where systems at pH 8 show longer emulsion stability. These results confirm the relevance of performing both interfacial tension and viscoelasticity measurements to select optimal conditions to assess emulsion stability, as well as the excellent potential of crayfish proteins as a food emulsifier. Thus, although the pH conditions studied were outside of the pH range that is normally used for food emulsions, in a previous paper (Romero, Cordobes, & Guerrero, 2009) it was found that, in order to enhance crayfish-based emulsion stability, it was convenient to perform the emulsification process at extreme pH conditions and then to modify the pH towards milder values. In any case, further research would be devoted to explore the typical range for food applications. Acknowledgements The authors acknowledge the financial support from the MEC (FPU grant). This work was also part of a research project No. AGL2007-65709 supported by the Spanish MCYT and FEDER and No. P09-TEP-4875 supported by the CICE (Andalousian Government, Spain).

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