Structural Characteristics of Hydrolysates of Proteins from Extracted

Biomacromolecules 2005, 6, 3137-3145

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Structural Characteristics of Hydrolysates of Proteins from Extracted Sunflower Flour at the Air-Water Interface Jose´ Minõnes Conde,†,‡ Juan M. Rodrı´guez Patino,*,† and Jose´ Minõnes Trillo‡ Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcıá Gonza´ lez, 1, 41012-Sevilla, and Departamento de Quı´mica Fı´sica, Facultad de Farmacia, Universidad de Santiago de Compostela Received July 4, 2005; Revised Manuscript Received August 24, 2005

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH ) 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 °C were determined from π-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the π-AAPPARENT isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis. Introduction A fundamental understanding of the physical, chemical, and functional properties of proteins and the changes these properties undergo during processing is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins, are to be increasingly used in traditional and processed food products. The functional properties of proteins in foods are related to their structural and other physicochemical characteristics.1 The precise role of protein structure and how its structural transformation in a food contributes to functional properties are not well understood and are the topic of much research.2-5 Plant proteins are being using successfully for the formation and stabilization of new food products,6-9 most of them presented commercially as dispersions (emulsions and foams). On the other hand, fat extraction from plant seeds yields a large amount of defatted meal, which, among other nutritional and nonnutritional components, contains a significant amount of proteins, polysaccharides, and fiber that could be used as food ingredients. Enzymatic hydrolysis is frequently used to improve the functional and nutritional properties of food proteins.6,7,10-12 The objective of this research program is to study the potentiality of biomacromolecules extracted from plants to produce new food formulations of nutritional and biological * To whom correspondence should be addressed. Phone: +34 95 4556446. Fax: +34 95 4556447. E-mail: [email protected]. † Universidad de Sevilla. ‡ Universidad de Santiago de Compostela.

interest. Plant proteins, including their hydrolysate and peptide derivatives, are of great importancesfrom the qualitative, quantitative, and economic points of views, in such formulations (health products in the forms of emulsions, foams, or nano- and microparticulate systems). In this work we complement a previous study13 by investigating the structural characterization and topography of spread films of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH)slow (5.62%), medium (23.5%), and high (46.3%)sat the air-water interface at pH 7 and 20 °C. Hydrolysates with a low degree of hydrolysis have improved functional properties (mainly foaming and emulsifying capacity),6,7,11,14,15 those with a variable degree of hydrolysis are used as flavorings,11 while extensive hydrolysates are used as nutritional supplements and in special medical diets (functional foods).16-20 The extended use of protein hydrolysates in traditional and new, processed “health” (safe, high-quality) food formulations requires extensive and systematic investigation of their surface properties, including static and dynamic conditions. However, as far as we know, the structural characteristics of SP hydrolysates at the air-water interface have not been analyzed so far. The protein film structure is important from a practical point of view because it defines its utility. From a fundamental point of view, orientational phenomena and domain structure are of particular interest. In this sense the structure and topography of the monolayer are of great utility. The film structural characteristics were determined from π-A isotherms coupled with Brewster angle microscopy (BAM), a technique that is sensitive, effective, and noninvasive.

10.1021/bm050469s CCC: $30.25 © 2005 American Chemical Society Published on Web 10/07/2005

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Table 1. Physicochemical Properties of Sunflower Protein Isolate component

composition (%)

moisture ash protein content soluble sugars polyphenols others

5.10 ( 0.83 3.00 ( 0.08 82.81 ( 0.45 0.22 ( 0.02 0.45 ( 0.08 8.60

Table 2. Solubility at pH 7 and Chemical Composition of Sunflower Protein Isolate and Protein Hydrolysates from Sunflower Protein Isolate at Different Degrees of Hydrolysis (DH)

isolate SPI hydrolysate at DH ) 5.62 SPI hydrolysate at DH ) 23.5 SPI hydrolysate at DH ) 46.3

protein (%, w/w)

modified protein

solubility (%, w/w) at pH 7.0

82.8 93.9 80.8 74.5

60.5 83.9 94.7

27.9 70.8 86.2 92.8

Materials and Methods Materials. The isolation of sunflower proteins (SPI) from defatted sunflower meal, the preparation of sunflower protein hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis (DH), the determination of solubility, and chemical characterization (including the determination of molecular weight by gel filtration chromatography, amino acid analysis by high-performance liquid chromatography, and chemical composition) have been described elsewhere.13 The physicochemical properties of SPI and the solubility and chemical composition of SPI and its hydrolysates are included in Tables 1 and 2, respectively.13 Proteins represent the main component of the isolate, but soluble sugars (0.22%) and polyphenols (0.45%) represent minor components. The protein concentration in the isolate and in protein hydrolysates is higher than in the original sunflower meal (33.3%, w/w)13 but decreases as the degree of hydrolysis increases. However, the modified protein increases with the degree of hydrolysis due to the action of the enzyme on native SPI. Solubility is lower for SPI than for its hydrolysates and increases with the degree of hydrolysis To form the spread surface film, protein was spread in the form of a solution using water at pH 7 as a spreading solvent. The sample was stored at 4 °C, and all work was done without further purification. Samples for interfacial characteristics of protein films were prepared using Milli-Q ultrapure water and were buffered at pH 7, using Trizma [(CH2OH)3CNH2/(CH2OH)3CNH3Cl] as supplied by Sigma (>95%) without further purification. Sodium azide (Sigma) was added (0.05 wt %) as an antimicrobial agent. The ionic strength was 0.05 M in all the experiments. Equilibrium Surface Pressure. Equilibrium spreading pressures (πe) of SPI and its hydrolysates at 20 °C were measured by the Wilhelmy plate method as described elsewhere.21,22 Briefly, the equilibrium spreading pressures were obtained by spreading various 50 µL aliquots of SPI and its hydrolysates in solution (0.96-1.24 mg/mL) on the

aqueous subphase and then sprinkling various amounts of the spray-dried protein on the aqueous solution. The reduction in surface tension (σ) was recorded continuously by a device connected to the tensiometer. Equilibrium was assumed when the surface tension did not change by more than 0.5 mN/m in 30 min. If no surface tension decrease was observed upon further addition of protein molecules, the final surface pressure value was taken as πe, where πe ) σo - σe; σo is the surface tension of the aqueous phase and σe is the surface tension at equilibrium. Some experiments were repeated (4 times) with different protein solutions. It was found that σe could be reproduced to (0.5 mN/m. Surface Film Balance. Measurements of the surface pressure (π) versus average trough area (A) were performed on a fully automated Langmuir-type film balance using a maximum area of 5.62 × 10-2 m2, as described elsewhere.23 The subphase temperature was controlled at 20 °C by water circulation from a thermostat, within an error range of (0.5 °C. The temperature was measured by a thermocouple located just below the air-water interface. Aliquots of aqueous solutions of protein fractions (0.961.24 mg/mL) at pH 7 were spread on the interface. To allow for spreading, adsorption, and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. The spreading method adopted in these experiments will require further discussion in the next section. The compression rate was 3.3 cm min-1, which is the highest value for which isotherms have been found to be reproducible in preliminary experiments with disordered24 and globular proteins.25 All isotherms were recorded continuously by a device connected to the film balance and then analyzed offline. Each isotherm was measured 5 times using new aliquots of aqueous solutions of proteins. The mean deviation was within (0.5 mN/m for surface pressure and (3 × 10-4 m2 for trough area. Brewster Angle Microscope (BAM). A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography of the monolayer. Further characteristics of the device and operational conditions were described elsewhere.23,24 The surface pressure measurements, area, and gray level as a function of time were carried out simultaneously by means of a device connected between the film balance and BAM. These measurements were performed during continuous compression and expansion of the monolayer at constant rate with different shutter speeds ranging from 1/50 to 1/500 s. In a previous work23 we have deduced that a shutter speed of 1/50 s is appropriate for the analysis of protein films at the air-water interface at low surface pressures, but higher shutter speeds (1/250 or 1/500 s) are more appropriate for the analysis of protein films at higher surface pressures. The reflectivity at each point in the BAM image depends on the local thickness and film optical properties. These parameters can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light.23,24 At Brewster angle,26 I ) Cδ2, where I is the reflectivity, C is a constant, and δ is the film thickness.

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Characteristics of Sunflower Protein Hydrolysates Table 3. Equilibrium Surface Pressure (πe) and Surface Pressure at the Transition between Structures I and II (πt) for a Sunflower Protein Isolate (SPI) and Its Hydrolysates at Different Degrees of Hydrolysis (DH) Spread Monolayers at the Air-Water Interface, at 20 °C and pH 7a

SPI SP hydrolysate at DH ) 5.62% SP hydrolysate at DH ) 23.5% SP hydrolysate at DH ) 46.3%

πe (mN/m)

πt (mN/m)

SA (mN/m)

23 29 22 23

12.8 14.7 14.3 12.3

>26.9 30.5 30.5 28.5

a The superficial activity of adsorbed films (SA) from ref 13 is included as reference.

Results and Discussion Spreading of SPI and Its Hydrolysates at the AirWater Interface. The equilibrium spreading pressure (πe) is the maximum surface pressure to which a spread monolayer may be compressed before monolayer collapse. Knowledge of πe is important because it represents the point at which spread monolayers become thermodynamically unstable with respect to the bulk liquid phase. The equilibrium spreading pressure is a measure of the surface activity of spread films at equilibrium. Spreading of proteins at fluid interfaces involves a surface solution process. The point at which this occurs and spreading commences depends on the particular protein structure.22,27 The spreading process is determined by the nature of protein-protein and proteinsubphase interactions; thus, πe must be a function of the degree of hydrolysis of the original protein. Equilibrium spreading pressures of SPI and its hydrolysates are shown in Table 3. The magnitude of πe was dependent on the protein fraction and on the degree of hydrolysis. The value of πe was minimal for SPI and its hydrolysates at higher degrees of hydrolysis (at DH 23.5% and 46.3%) and was a maximum for SP hydrolysate at DH 5.62%. The πe values for SPI and its hydrolysates at DH 23.5% and 46.3% were similar as those for a disordered protein.22 However, the value of πe for SP hydrolysate at DH 5.62% was similar as those for globular proteins from milk22 and soy.27 The minimum πe values for SPI, as compared to πe for SP hydrolysate at DH 5.62%, can be explained by the fact that the protein is more difficult to convert into a monolayer in the native form. That is, SP hydrolysate at DH 5.62% (with the highest πe value) may be spread more easily on aqueous solutions than the native SPI, as a consequence of changes in molecular conformation and in the flexibility of the molecule compared to those of native SPI. On the other hand, SP hydrolysate at DH 5.62% (with the highest πe value) may be spread more easily on aqueous solutions than hydrolysates at higher degrees of hydrolysis (with lower πe values). At higher degrees of hydrolysis, although protein solubility is at a maximum (Table 2), there is also some degree of aggregation, as will be discussed in the following sections. This interfacial aggregation may limit the spreading of the protein at the interface.27 Finally, the values of πe for SPI and its hydrolysates are lower than the superficial activity (SA) for adsorbed films13 (SA is the maximum surface pressure allowed during the protein adsorption from aqueous solutions). This means

Figure 1. Spreading of a sunflower protein isolate (SPI) at the airwater interface. (A) Surface pressure-trough area isotherm for an SPI monolayer spread at the air-water interface, during a compression-expansion cycle. (B) Surface pressure-apparent area isotherm for an SPI monolayer spread at the air-water interface, during a compression-expansion cycle. Temperature, 20 °C; pH 7; I ) 0.05 M. The equilibrium surface pressure (πe) and the surface pressure at the transition between structures I and II (πt) are indicated by arrows.

that films of SPI and its hydrolysates adopted different structures as they were spread or adsorbed at the air-water interface. Structural and Topographical Characteristics of Spread Monolayers of a Sunflower Protein Isolate. The π-trough area isotherm for an SPI spread monolayers at the air-water interface at pH 7 and 20 °C is shown in Figure 1A. By assuming that all spread protein molecules in SPI (Table 2) form the monolayer and no loss into the bulk aqueous phase takes place, a π-AAPPARENT isotherm was plotted (Figure 1B). This π-AAPPARENT isotherm shows an unusual mass area as compared with those of typical π-A isotherms for proteins.24,25,28,29 It can be seen that the π-AAPPARENT isotherm appeared at low mass areas. Thus, the extrapolated area in the more condensed structure of the SPI monolayer is lower than 1 m2/mg, which is a typical value for well-spread protein monolayers at the air-water interface.30 These experiments were repeated at the same waiting time after the protein spreading for a undisturbed monolayer. The π-AAPPARENT isotherms obtained under these conditions are practically the same (within the experimental error) as those obtained after consecutives compression-expansion cycles of the same monolayer. On the other hand, the rate of monolayer compression did not have a significant effect on the π-AAPPARENT isotherm (data not shown), and the isotherms were reproducible after repeated experiments for different aging times using new aliquots of the protein spreading solution. Different criteria for quantitative spreading of SPI monolayers were adopted in this work. First, to allow the quantitative adsorption of the protein on the interface the

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Figure 2. Visualization of SPI monolayers spread at the air-water interface at pH 7 and 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 mm, and the vertical direction corresponds to 470 mm. Surface pressure (mN/m): (a) 0.6, (b) 3, (c) 15, (d) 20.4, (e) 26, and (f) 30.

monolayer was not under any surface pressure during the spreading process. Second, the best way to check the efficiency of spreading is to use several spreading solutions of different protein concentrations.30 The results of these tests confirm the quantitative spreading of SPI monolayers on the air-water interface. Finally, the method used by Trurnit31 gave better results for the quantitative spreading of SPI, as was observed for WPI,25 BSA,32 β-lactoglobulin,33 and soy protein28,29 monolayers. These results demonstrate that, although the π-AAPPARENT isotherm is not a thermodynamic description of the state of an SPI monolayer, it is possible to measure reproducible π-AAPPARENT isotherms for SPI monolayers with the protocol adopted in this work, as discussed elsewhere.29 Thus, the structure that SPI molecules adopt at the air-water interface can be deduced from these π-AAPPARENT isotherms. The results of the π-AAPPARENT isotherms (Figure 1B), together with the compressional coefficient deduced from the slope of the π-AAPPARENT isotherm (κ ) -dπ/dA), indicate that an SPI monolayer at the air-water interface adopts two different structures or condensation states and the collapsed phase. The surface pressure at the transition or at the change in condensation state of the monolayer (πt) is included in Table 3. At low surface pressures (π < πt) SPI may exist34-36 as trains with all amino acid segments located at the interface or may adopt a loop conformation (structure I). At higher surface pressures (π > πt), and up to the equilibrium surface pressure, amino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails (structure II). The more condensed conformation at higher surface pressures (structure II) is displaced toward the bulk phase at the collapse point.34 The collapse was produced at a surface pressure higher than the equilibrium surface pressure for SPI (which is included in Figure 1 by means of an arrow), a phenomenon which indicates that the monolayer was in a metastable state at π > πe . Results of BAM, in particular BAM images (Figure 2) and the reflectivity (Figure 3) as a function of time and/or surface pressure obtained with SPI monolayers clearly show the same structural characteristics as those deduced from the π-AAPPARENT isotherm. The reflection of the interface

illuminated with p-polarized light at the Brewster angle for the SPI-covered interface is no different from the background of the clean subphase. BAM images corroborate that only a homogeneous phase is present during the compression of SPI up to the monolayer collapse. That is, from the BAM images it is impossible to distinguish between different structures that residues of SPI molecules adopt at the airwater interface. In fact, BAM images for SPI monolayers with structure I (Figure 2b), near the monolayer transition between structures I and II (Figure 2c), and with structure II (Figure 2d) are practically indistinguishable. The domains appear to be of uniform reflectivity, suggesting homogeneity in thickness and isotropy in the plane vertical to the molecular chain. The images are practically independent of the analyzer angle (data not shown), suggesting that SPI residues spread at the air-water interface have the same isotropy in the plane vertical to the molecular chain (that is, no crystalline domains are formed), no matter what the structure adopted by SPI residues at the interface. BAM images for a collapsed film (Figure 2, parts e and f) are practically the same as for structures I and II. However, at the collapse point some folds (which are indicated by arrows in Figure 2, parts e and f) are observed along the interface with different illumination. Differences in the image contrast are an indication that collapsed residues of SPI monolayers are aligned, on average, parallel to the barrier movement, a typical behavior of small and large molecules under extensional flow.37-39 Finally, after the expansion, the monolayer undergoes break up of the collapsed structure up to a two-dimensional foam structure (Figure 2a), as the surface pressure approaches zero. This structure is also present during next compressions at π ≈ 0. In this regards the topography of SPI at the air-water interface is similar to that for milk proteins.24 Time evolution of surface pressure and reflectivity (I) during the compression-expansion cycle of SPI monolayers spread on water is shown in Figure 3A. It can be seen that I increases as the monolayer is compressed, passes through a maximum at the collapse point, and then decreases with monolayer expansion. Upon compression, in the expanded monolayer region, the reflectivity (I) is practically constant (3 × 10-7 to 8 × 10-7 au) with dust or protein aggregates (generated in previous compressions) causing some peaks

Characteristics of Sunflower Protein Hydrolysates


Figure 4. Surface pressure-apparent area isotherm (only during the monolayer compression) for spread monolayers at the air-water interface of an SP hydrolysate at DH ) 5.62%. Temperature, 20 °C; pH 7; I ) 0.05 M. The equilibrium surface pressure (πe) and the surface pressure at the transition between structures I and II (πt) are indicated by arrows. Symbols: (O) spreading of 0.144 mg and 30 min after spreading, spreading 0.096 mg at (]) 30 min after spreading, (4) 60 min after spreading, and (g) 90 min after spreading.

Figure 3. (A) Time evolution of the (O) reflectivity and (/) surface pressure upon a compression-expansion cycle for an SPI monolayer spread at the air-water interface (third compression). (B) Time evolution of the (O) monolayer thickness and (/) surface pressure upon a compression-expansion cycle for an SPI monolayer spread at the air-water interface. (C) The effect of surface pressure on the monolayer thickness. Temperature, 20 °C; pH 7; I ) 0.05 M; shutter speed, 1/250 s; monolayer aging, 60 min. The same topography was observed for different shutter speeds and monolayer aging.

in the reflectivity curve. The reflectivity increases abruptly during the monolayer compression (near the transition between structures I and II, at π = 10 mN/m), reaching a value of ca. 5.5 × 10-6 au at the equilibrium surface pressure, in the condensed regime (with a structure II), and even higher at the collapse. During the monolayer expansion, similar phenomena are observed. However, some reflectivity peaks of pronounced intensity are present on expansion even at the maximum molecular area. Thus, it is evident that the small nuclei of collapsed SPI, which are created during monolayer compression at π > πe, are maintained during

expansion, leading to similar intense reflectivity peaks (during both phases of the cycle). At a microscopic level, the compression-expansion cycle was practically reversible within the time of the experiment because the I-time curve during compression was practically the same as that during expansion (Figure 3A). The increase in reflected light intensity with surface pressure (Figure 3A), and especially at the monolayer collapse, suggests an increase in the monolayer thickness from a more expanded to a more condensed structure, followed by a further increase as monolayer collapse takes place (Figure 3B). As for most protein monolayers,36 reflectivity did not depend on the method adopted for the spreading of SPI, but only depended on the surface pressure (data not shown). These results corroborate the idea23,24,28,29 that the I-π curve could reflect the surface equation of state of the spread material at the air-water interface. This master curve is characteristic for any material spread at the airwater interface. That is, the surface pressure is a fundamental variable that defines the degree of interaction (i.e., the monolayer structure) between the film forming components at the air-water interface. From the I-π curve we can obtain the evolution of the monolayer thickness with surface pressure (Figure 3C). In fact, at the end of the monolayer compression with structure I (at πt), the monolayer thickness was δ ) 1.7 ( 0.2 nm. The monolayer thickness increased during compression with structure II, and at πe (with the maximum monolayer condensation before the collapse), the value of δ (δπe ) 5.2 ( 0.3 nm) was 3 times higher than in the more condensed state of the monolayer with structure I. Finally, at the highest surface pressure δ ) 14.7 ( 0.5 nm, which means that the monolayer thickness increased 2.8 times in relation to that at πe. These results confirm that a multilayer formation takes place at the highest surface pressure at the monolayer collapse point. Structural and Topographical Characteristics of Spread Monolayers of SP Hydrolysates. The π-AAPPARENT isotherms for spread monolayers at the air-water interface of SP hydrolysates at DH ) 5.62%, 23.5%, and 46.3%, at pH 7 and 20 °C are shown in Figures 4-6, respectively. These

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Figure 5. Surface pressure-apparent area isotherm for spread monolayers at the air-water interface of an SP hydrolysate at DH ) 23.5%, during a compression-expansion cycle. Temperature, 20 °C; pH 7; I ) 0.05 M. The equilibrium surface pressure (πe) and the surface pressure at the transition between structures I and II (πt) are indicated by arrows.

Figure 6. Surface pressure-apparent area isotherm for spread monolayers at the air-water interface of an SP hydrolysate at DH ) 46.3%. (A) Aqueous subphase at pH 7 (only during the monolayer compression). Different lines are for different aging times and different spreading aliquots. (B) Aqueous subphase at pH 7 and NaCl ) 0.5 M, during different compression-expansion cycles at different aging times (min): (O) 30, (4) 90, and (]) 780. Temperature, 20 °C; I ) 0.05 M. The equilibrium surface pressure (πe) and the surface pressure at the transition between structures I and II (πt) are indicated by arrows.

isotherms were plotted assuming that all spread protein molecules in the hydrolysate (Table 2) form the monolayer and no loss into the bulk aqueous phase takes place. There was a difference in the π-AAPPARENT isotherms as a function of the degree of hydrolysis. It can be seen that there is a shift of the isotherms toward lower mass areas as the degree of hydrolysis increases. That is, adopting the Trurnit method and optimizing the amount of protein spreading, the surface pressure at the maximum area was equal to zero, although the π-AAPPARENT isotherms were displaced toward low mass areas, which could be an indication of a protein loss into the bulk phase. But this protein loss, probably in the form of aggregates, does not affect the reproducibility of the

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π-AAPPARENT isotherms, as was observed for different compressions and at different aging times for SP hydrolysates at DH ) 5.62% (Figure 4) and at DH ) 46.3% (Figure 6). However, major spreading problems were observed for SP hydrolysate at DH ) 46.3%, because the surface pressure at the end of the compression (at the lowest mass apparent area) was lower than πe (Figure 6A). For larger amounts of spread protein the surface pressure at the maximum trough area was not equal to zero, which is a requirement for quantitative spreading of proteins.30 The spreading of SP hydrolysate at DH ) 46.3% was improved on aqueous solutions at high ionic strength. In fact, we can see that for SP hydrolysate at DH ) 46.3% spread monolayers on aqueous solutions at 0.5 M NaCl the π-AAPPARENT isotherms were displaced toward higher mass area and that the monolayer collapsed at a surface pressure higher than the value of πe (Figure 6B). There was a difference in the π-AAPPARENT isotherms as a function of time after protein spreading. It can be seen that there was a shift of the isotherms toward higher mass areas as the spreading time increased. This behavior is opposite to that observed for nonquantitative protein spreading.30,33 The results in Figure 6B indicate that the structural characteristics of spread monolayers of SP hydrolysate at DH ) 46.3% depend on film aging. That is, the protein film changed significantly over time, which is a characteristic behavior of globular milk40 and soy28,29 proteins spread at the air-water interface. On the other hand, the results in Figure 6B also shown that the π-AAPPARENT isotherms registered after different aging times were parallel to each other. In addition, in Figure 6B we also observed that the maximum surface pressure also increased with the aging time. Finally, at the higher aging time (after the third compression at 780 min) the π-AAPPARENT isotherms were reproducible after repeated compressions (data not shown). These results demonstrate that the aging effect observed in SP hydrolysate at DH ) 46.3% monolayers spread at the air-water interface is due to unfolding of the protein at the interface.41,42 However, we do not reject the possibility that a diffusional loss of aggregated protein takes place during the spreading process, which afterward is incorporated at the interface.28,29 In the absencesas for SP hydrolysate at DH ) 5.62% (Figure 4) and DH ) 23.5% (Figure 5)sor in the presences as for SP hydrolysate at DH ) 46.3% spread on aqueous solutions of NaCl (Figure 6B at long time)sof an aging time, the π-AAPPARENT isotherms were reproducible. Thus, although the π-AAPPARENT isotherm is not a thermodynamic description of the state of a spread monolayer for SP hydrolysates, the monolayer structure can be deduced from these π-AAPPARENT isotherms. That is, the monolayer structure depends on the interactions between the molecules at the interface, which is reflected by the surface pressure. From these isotherms and with the help of the compressional coefficient we can deduce that SP hydrolysates spread at the air-water interface adopt two different structures (Figures 4-6). The surface pressure at the transition between structures I and II decreased with the degree of hydrolysis (Table 3).

Characteristics of Sunflower Protein Hydrolysates

Figure 7. Visualization of SP hydrolysate at DH ) 5.62% monolayers spread at the air-water interface at pH 7 and 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Surface pressure (mN/m): (a) 0.1, (b) 5.7, (c) 11.4, and (d) 24.4.

Figure 8. Visualization of SP hydrolysate at DH ) 23.5% monolayers spread at the air-water interface at pH 7 and 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Surface pressure (mN/m): (a) 3.4, (b) 13.0, (c) 17.3, and (d) 21.5.

The monolayer topography (BAM image) clearly shows significant differences between monolayers of SP hydrolysates as a function of degree of hydrolysis (Figures 7-9). The topography of monolayers of SP hydrolysate at DH ) 5.62% (Figure 7) shows that the domains that residues of protein molecules adopt at the air-water interface at low surfaces pressures appeared to be of uniform reflectivity (Figure 7a), suggesting homogeneity in thickness and film isotropy. After the monolayer compression with a structure I different domains of high intensity were observed at the interface (Figure 7b), which increased in number up to the end of the monolayer compression with structure I (Figure 7c). At the collapse point, the intensity increased and aggregates of collapsed protein residues were observed at the interface (Figure 7d). That is, by BAM we are able to distinguish between different structures or states of condensation in protein monolayers for SP hydrolysate at DH ) 5.62%. BAM images for monolayers of SP hydrolysate at DH ) 23.5% (Figure 8) corroborated the fact that a heterogeneous


phase was present during the compression, with numerous microdomains that increased in brightness as the monolayer was compressed (Figure 8a-c) up to the collapse (Figure 8d). The microdomains for an SP hydrolysate at DH ) 23.5% monolayer were smaller and the monolayer segregation was higher than for an SP hydrolysate at DH ) 5.62% monolayer. At π > πe some fractures of collapsed SP hydrolysate at DH ) 23.5% were observed over the interface from differences in illumination (Figure 8d), as the monolayer cracks cooperatively over large length scales giving some regions of collapsed proteins separated by other regions with a lower condensation state. During the expansion phase the same features as those in the compression phase were observed (data not shown), which demonstrate the reversibility of the compression-expansion cycle at a microscopic level. Finally, at the lower surface pressures (at π = 0) after the expansion, the presence of two-dimensional foams (data not shown) was the typical feature of SP hydrolysate monolayers (at DH ) 23.5%) at the air-water interface. SP hydrolysate at DH ) 46.3% (Figure 9) presented the same features during compression up to the monolayer collapse (Figure 9, parts b and c) as SP hydrolysate at DH ) 23.5% (Figure 8) and the presence of two-dimensional foams during monolayer expansion at π = 0, or at the beginning of compression of a previously compressed monolayer, at π = 0 (Figure 9a). Thus, the protein aggregates formed during monolayer compression were present at the interface during successive compression-expansion cycles. From the I-π curves, which reflect the nanoscopic surface equation of state of the spread material at the air-water interface (data not shown), we can obtain the evolution of the monolayer thickness with the surface pressure (Figure 10). The thickness increased as the monolayer was compressed and was a maximum at the monolayer collapse. That is, an increase in monolayer thickness with surface pressure was produced, from more expanded (structure I) to more condensed (structure II) structure, followed by a further increase at the monolayer collapse. The presence of some fractures of collapsed SP hydrolysate at DH ) 23.5% and at π > πe, giving some regions of collapsed proteins separated by other regions with a lower condensation state (Figure 8d), was reflected by the abrupt drops in monolayer thickness near to the collapse (Figure 10B). Interestingly, at the more expanded structure (with structure I) and at the same surface pressure the monolayer thickness was practically the same no matter what the degree of hydrolysis. However, in the most condensed structure, at the monolayer collapse, the monolayer thickness was higher for SP hydrolysates with lower degrees of hydrolysis (Figure 10). In fact, as the molecular weight of the protein fractions decreased at higher degrees of hydrolysis, as a consequence of the enzymatic treatment,13 the interactions between protein domains were lower and the monolayer thickness decreased. In addition, the higher aggregation between protein microdomains in SP hydrolysate at DH ) 46.5% (Figure 9, parts b and c) produced high noise peaks in the monolayer thickness (Figure 10C). That is, a more heterogeneous segregated monolayer was produced at the highest degree of hydrolysis.

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Figure 9. Visualization of SP hydrolysate at DH ) 46.3% monolayers spread at the air-water interface at pH 7 and 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Surface pressure (mN/m): (a) 0, (b) 13.9, and (c) 10.

Figure 10. The evolution of monolayer thickness with surface pressure upon compression for monolayers of SP hydrolysates spread at the air-water interface. Degree of hydrolysis: (A) 5.62%, (B) 23.5%, and (C) 46.3%. Temperature, 20 °C; pH 7; I ) 0.05 M.

Conclusions In this work we have used a surface film balance and Brewster angle microscopy to analyze the structural characteristics (structure, topography, interfacial reflectivity, and monolayer thickness) of hydrolysates from sunflower protein isolate (SPI) spread films on the air-water interface. An SPI monolayer at the air-water interface adopts two different structures or condensation states (structures I and II) and the collapse phase. This work demonstrates that the structural and topographical characteristics of spread monolayers of SP hydrolysates at the air-water interface depend on the degree of hydrolysis. The surface pressure at the transition

between structures I and II for spread monolayers of SP hydrolysates at the air-water decreases with the degree of hydrolysis. Monolayer topography (BAM image, reflectivity, and thickness) confirms that there exist significant differences between monolayers of SP hydrolysates as a function of the degree of hydrolysis. The protein microdomains and their brightness increase as the monolayer is compressed up to the collapse. The microdomains at the air-water interface for SP hydrolysates are smaller, and the monolayer segregation is higher, as the degree of hydrolysis increases. The monolayer thickness increases with the surface pressure from a more expanded (structure I) to a more condensed structure (structure II), and a further increase takes place as the monolayer collapses. The monolayer thickness is higher for SPI than for its hydrolysates. At the collapse point the monolayer thickness is higher for SP hydrolysates with lower degrees of hydrolysis. The spreading problems observed for SP hydrolysate at DH ) 46.3% (Figure 6A), in addition to the segregation of protein microdomains (which does not favor the formation of a gel-like monolayer at the higher surface pressures) (Figure 9) and the low monolayer thickness (Figure 10), have negative effects on the stabilization of the interfacial film. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis. The inclusion of phospholipids (due to their intrinsic nutritional and therapeutic properties) in addition to SP hydrolysates can overcome the problems derived from an interfacial film with low protein-protein interactions (with low mechanical properties), as the small peptides produced during enzymatic hydrolysis dominate the interface at the higher degrees of hydrolysis. This work is underway at present. Acknowledgment. The authors acknowledge the support of CICYT though Grants AGL2001-3843-C02-01 and AGL2004-01306/ALI. Drs. F. Millań and J. J. Pedroche (Instituto de la Grasa, C.S.I.C., Seville, Spain) are thanked for performing the preparation and characterization of the sunflower protein isolate and its hydrolysates and for useful discussion concerning this project. References and Notes (1) Damodaran, S. In Food Proteins and their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997; p. 1. (2) Dickinson, E. An Introduction to Food Colloids; Oxford University Press: Oxford, U.K., 1995. (3) Dickinson, E.; McClements, D. J. AdVances in Food Colloids; Blackie: Glasgow, 1999. (4) Dickinson, E. Colloids Surf., B 1999, 15, 161. (5) Horne, D. S.; Rodrı´guez Patino, J. M. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; p 857.

Characteristics of Sunflower Protein Hydrolysates (6) Damodaran, S.; Paraf. A. Food Proteins and their Application; Dekker: New York, 1997. (7) Kilara, A.; Panyam, D. Crit. ReV. Food Sci. Nutr. 2003, 43, 607. (8) O ¨ rnebro, J.; Nylander, T.; Eliasson, A.-C. J. Cereal Sci. 2000, 31, 195. (9) Kinsella, J. E. J. Am. Oil Chem. Soc. 1979, 56, 242. (10) Vioque, J.; Sańchez-Vioque, R.; Clemente, A.; Pedroche, J.; Millań, F. J Am. Oil Chem Soc. 1999, 76, 819. (11) Vioque, J.; Clemente, A.; Pedroche, J.; Yust, M. M.; Millań, F. Grasas Aceites 2001, 52, 132. (12) Pedroche, J.; Yust, M. M.; Lqari, H.; Giroń-Calle, J.; Alaiz, M.; Vioque, J.; Millań, F. Food Chem. 2004, 88, 337. (13) Minõnes Conde, J.; Yust, M. M.; Pedroche, J. J.; Millań, F. R.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 2005, 53, 8038. (14) Lahl, W. J.; Braun, S. D. Food Technol. 1994, 48, 68. (15) Foegeding, E. A.; Davis, J. P.; Doucet, D.; McGuffey, M. K. Trends Food Sci. Technol. 2002, 13, 151. (16) Panyam, D.; Kilara, A. Trends Food Sci. Technol. 1996, 7, 120. (17) Cordle, C. T. Food Technol. 1994, 48, 72. (18) Kawamura-Yusui, N.; Kaito, M.; Nakagawa, N.; Fugita, N.; Ikowa, I.; Gabazza, E. C. J. Clin. Lab. Anal. 1999, 13, 31. (19) Ziegler, F.; Ollivier, J. M.; Cynober, L.; Masinim, J. P.; CodrayLucas, C.; Levis, E.; Giboudeau, J. Gut 1990, 31, 1277. (20) Clare, D. A.; Swaisgood, H. E. J. Dairy Sci. 2000, 83, 1187. (21) Rodrı´guez Ninõ, M. R.; Carrera, C.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 161. (22) Rodrı´guez Ninõ, M. R.; Carrera, C.; Rodrı´guez Patino, J. M. J. Am. Oil Chem. Soc. 2001, 78, 873. (23) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 2484. (24) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Food Hydrocolloids 1999, 13, 401.

Biomacromolecules, Vol. 6, No. 6, 2005 3145 (25) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (26) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1992. (27) Molina, S. E.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Anõń, C.; Rodrı´guez Patino, J. M. Colloids Surf., B 2003, 32, 57. (28) Carrera, C.; Molina, S.; Rodrı´guez Ninõ, M. R.; Anõń, C.; Rodrı´guez Patino, J. M. Langmuir 2003, 19, 7478. (29) Carrera, C.; Molina, S.; Rodrı´guez Ninõ, M. R.; Anõń, C.; Rodrı´guez Patino, J. M. Food Hydrocolloids 2004, 18, 335. (30) MacRitchie, F. AdV. Colloid Interface Sci. 1986, 25, 345. (31) Trurnit, H. J. J. Colloid Sci. 1960, 15, 1. (32) Cho, D.; Frances, E. I.; Narshimhan, G. Colloids Surf., A 1996, 117, 45. (33) Kra¨gel, J.; Grigoriev, D. O.; Makievski, A. V.; Miller, R.; Fainerman, V. B.; Wilde, P. J.; Wu¨stneck, R. Colloids Surf., B 1999, 12, 391. (34) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (35) Benjamins, J. Static and Dynamic Properties of Protein Adsorbed at Liquid Interfaces. Ph.D. Thesis, Wageningen University, 2000. (36) Rodrı´guez Ninõ, M. R.; Carrera, C.; Pizones, V.; Rodrı´guez Patino, J. M. Food Hydrocolloids 2005, 19, 417. (37) Rodrı´guez Patino, J. M.; Carrera, C. Langmuir 2004, 20, 4530. (38) Carrera, C.; Rodrı´guez Patino, J. M. Langmuir 2004, 20, 6327. (39) Rodrı´guez Patino, J. M.; Carrera, C. Biomacromolecules 2004, 5, 2065. (40) Garofalakis, G.; Murray, B. S. Colloids Surf., B 1999, 12, 231. (41) Tronin, A.; Dubrovsky, T.; Dubrovskaya, S.; Radicchi, G.; Nicolini, C. Langmuir 1996, 12, 3272. (42) Dickinson, E.; Matsumura, Y. Int. J. Biol. Macromol. 1991, 13, 26.

BM050469S

Structural Characteristics of Hydrolysates of Proteins from Extracted

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