Pectin Coacervates Studied by Small

Dec 23, 2006 - Small-angle neutron scattering (SANS) has been used to investigate the microstructure of β-lactoglobulin/pectin coacervates prepared b...
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J. Phys. Chem. B 2007, 111, 515-520

515

Microstructure of β-Lactoglobulin/Pectin Coacervates Studied by Small-Angle Neutron Scattering Xiaoyong Wang,† Yunqi Li,†,‡ Yu-Wen Wang,† Jyotsana Lal,§ and Qingrong Huang*,† Department of Food Science, Rutgers UniVersity, 65 Dudley Road, New Brunswick, New Jersey 08901, State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China, and Intense Pulsed Neutron Source DiVision, Argonne National Laboratory, Argonne, Illinois 60439 ReceiVed: May 29, 2006; In Final Form: October 31, 2006

Small-angle neutron scattering (SANS) has been used to investigate the microstructure of β-lactoglobulin/ pectin coacervates prepared by different initial protein/polysaccharide weight ratio (r), sodium chloride concentration (CNaCl), and pectin charge density. The higher r and higher pectin charge density lead to higher scattering intensity at small q range (0.007 Å-1 < q < 0.02 Å-1), suggesting that the charges of pectin chains are screened significantly by the binding of oppositely charged protein molecules, leading to a tighter aggregation of pectin chains. On the other hand, the appearance of a shoulder peak at intermediate q range (0.04 Å-1 < q < 0.2 Å-1) is used to interpret the formation of protein domains in β-lactoglobulin/pectin coacervates. At CNaCl ) 0.1 M, the coacervate of β-lactoglobulin and pectin A does not show a shoulder peak at intermediate q range at r ) 10:1, suggesting that protein molecules are separately bound on pectin chains. However, a shoulder peak appears at intermediate q range at r ) 20:1 and 30:1, and the average protein domain size estimated from the shoulder peak position is 7.2 and 8.5 nm, respectively, for these two coacervates. When CNaCl increases from 0.05 to 0.2 M, the shoulder peak shifts toward smaller q and becomes broader, indicating that the addition of a higher amount of salt leads to a more heterogeneous coacervate structure. Pectin B with a lower linear charge density favors the formation of larger protein domains. The formation of protein domains in β-lactoglobulin/pectin coacervates is partially ascribed to the self-aggregation of β-lactoglobulin molecules. Two kinds of microstructures of β-lactoglobulin/pectin coacervates with and without observable protein domains have been proposed.

Introduction Coacervation has attracted increasing interest in the past two decades because of its implication in many biological processes like the organization of living cell1 and its use in many industrial applications such as microencapsulation,2 protein separation and purification,3 and processed foods.4 Coacervation is a phenomenon in which a macromolecular aqueous solution separates into two immiscible liquid phases.5 The denser phase is called coacervate, which is relatively concentrated in macromolecules and is in equilibrium with the relatively dilute macromolecular liquid phase. The general picture for protein/polysaccharide coacervation from previous studies6,7 is that protein molecules initially bind on polysaccharide chains to form primary soluble protein/polysaccharide complexes at the first critical pH (pHc), even when protein molecules carry the same net charges as polysaccharide chains because of the heterogeneous distribution of protein charges.8,9 At the second critical pH (pHφ), which is usually below the protein isoelectric point, strong electrostatic interaction between positively charged protein molecules and anionic polysaccharide chains will cause soluble protein/ polysaccharide complexes to aggregate into insoluble protein/ polysaccharide complexes, which ultimately settle at the bottom of the solution to generate the dense coacervate phase. For * Author to whom correspondence should be addressed. Phone: 732932-7193. Fax: 732-932-6776. E-mail: [email protected]. † Rutgers University. ‡ Chinese Academy of Sciences. § Argonne National Laboratory.

negatively charged weak acid (e.g., carboxylic acid)-based polysaccharides like pectin, with the decrease of pH below its pKa (∼3.0 for pectin10), due to the low charges of polysaccharide chains as well as the repulsion between the positively charged proteins, protein (e.g., β-lactoglobulin)/polysaccharide (e.g., pectin) coacervates may dissociate into soluble complexes, or even uninteracted protein molecules and polysaccharide chains.11 Because the coacervates formed by protein molecules and oppositely charged polysaccharide chains are mainly driven by the long-range character of the electrostatic interaction, physicochemical parameters, such as pH, ionic strength, polysaccharide linear charge density, protein surface charge density, rigidity of the polysaccharide chain, and protein/polysaccharide weight ratio, have been demonstrated to strongly influence the formation of protein/polysaccharide coacervates.12 Although the protein/polysaccharide coacervation has been widely studied, most of the previous works are mainly focused on the understanding of the phase boundary of coacervate formation, and the knowledge of the structure of protein/ polysaccharide coacervate is still quite lacking.13,14 Using confocal scanning laser microscopy, Sanchez et al.15,16 found that the internal structure of β-lactoglobulin/gum arabic coacervates was vescicular to sponge-like, exhibiting numerous spherical inclusions of water depending on the initial mixing ratio. Weinbreck et al.17 found that whey protein/gum arabic coacervate was the most concentrated and had the largest coacervate yield at protein/polysaccharide weight ratio of 2:1 and pH 4.0. Their small-angle X-ray scattering measurements

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Figure 1. The typical chemical structure of pectin.

suggested that the coacervate phase was dense and structured, and could be tuned by pH, protein/polysaccharide weight ratio, and ionic strength. In another work,18 Weinbreck et al. demonstrated that the diffusion of whey protein and gum arabic within the coacervate phase was reduced compared to that of the original dilute biopolymer mixture. Moreover, whey protein molecules were found to diffuse ten times faster than gum arabic molecules based on the results of fluorescence recovery after photobleaching. These very recent works provide important insight into the microstructure of protein/polysaccharide coacervates. However, the arrangement of protein molecules and polysaccharide chains in protein/polysaccharide coacervates remains unclear, and more work needs to be done to further disclose the microstructure of protein/polysaccharide coacervates. In this paper, we focus on the study of the microstructure of coacervates formed by β-lactoglobulin and two kinds of pectins with different linear charge densities using small-angle neutron scattering (SANS). β-Lactoglobulin is a model globular protein with a well-known structure.19,20 The monomer of β-lactoglobulin consists of 162 amino acids with two disulfide bonds and one free cysteine group. At normal physiological pH, β-lactoglobulin mainly exists in the dimer form with a molecular weight of 36 400 Da and a pH isoelectric point of around 5.2. The anionic polysaccharide examined in this work is pectin, which is a natural polymer extracted from plant cell walls. Pectin is used broadly not only in the food industry but also in the field of cosmetics and pharmaceutical applications.21,22 The typical chemical structure of pectin is shown in Figure 1. The major constituent common to pectin is a backbone chain structure of 1,4-linked R-D-galacturonic acid units interrupted by the 1,2linked L-rhamnopyranosyl residues in adjacent or alternate positions.23 Depending on the amount of methyl ester group above or below 50%, pectin is classified as high methoxyl (HM) pectin and low methoxyl (LM) pectin, respectively. In this work, the microstructures of coacervates formed by β-lactoglobulin with two low methoxyl pectins (pectin A and pectin B) carrying different amounts of galacturonic residues prepared at various initial protein/polysaccharide weight ratios (r) and sodium chloride concentrations (CNaCl) have been investigated by SANS. Experimental Section Materials. β-Lactoglobulin (lot JE 003-3-922) was obtained from Davisco Foods International, Inc. (Le Sueur, MN) and used without further purification. The powder composition was the following (g/100 g of powder): 5.2% moisture, 92.0% protein, 0.3% fat, and 2.5% ash. The β-lactoglobulin powder contains the genetic variants A and B in a nearly 1:1 ratio. Two pectin samples (A and B) were supplied by Danisco A/S, Denmark. Pectin A has 69% galacturonic residues, while there are 50% galacturonic residues in pectin B. These pectin samples were purified by dialysis (Spectra/Por dialysis membrane with molecular weight cutoff equal to 12 000), followed by freezedrying. The average molecular weights (Mn) of both purified pectins were about 7.0 × 105, as determined by gel permeation chromatography. Deuterium oxide (D2O, 99.9 atom % D) was purchased from Aldrich Chemicals (Milwaukee, MI). Sodium chloride (NaCl, purity >99%) and standard hydrochloric acid

(HCl, 0.5 N) were purchased from Fisher Scientific (Pittsburgh, PA). Milli-Q water was used in all experiments. Preparation and Compositional Analysis of β-Lactoglobulin/Pectin Coacervates. H2O is directly used as solvent in the preparation of all β-lactoglobulin/pectin coacervates. The stock solutions of β-lactoglobulin and pectin were mixed together with defined initial protein/polysaccharide weight ratio (r) and sodium chloride concentration (CNaCl). The final pectin concentration is fixed at 0.1 wt % for all β-lactoglobulin/pectin mixtures. A 0.5 N standard HCl solution was used to adjust the pH of the mixtures to 4.0. After acidification, the coacervates were collected after removal of the supernatant through centrifugation at 3000 rpm for 30 min. The yield of β-lactoglobulin/ pectin coacervate varies from 3% to 7%. It should be pointed out that r, CNaCl, and pH are the sample preparation conditions from which the coacervates are formed. The composition of β-lactoglobulin/pectin coacervates was analyzed by using the method similar to the previously published procedures.17,24 The water amount in the coacervates was determined at least twice by dry weighting. To determine the concentrations of β-lactoglobulin and pectin in the coacervates, β-lactoglobulin/pectin coacervates were first dissolved in sodium phosphate buffer of pH 7.4. Then, the contents of β-lactoglobulin and pectin were determined by using the size exclusion chromatography (SEC) system (DIONEX ultimate 3000) connected with a ZORBAX GF-450 gel filtration column and a UV detector, with the absorbance measured at 280 and 214 nm, respectively. The contents of β-lactoglobulin and pectin in the coacervates were finally calculated according to β-lactoglobulin and pectin calibration curves. For comparison, β-lactoglobulin content was also determined by UV spectrophotometer (Cary Eclipse) with the absorbance measured at 280 nm, and the results were comparable with SEC measurements. Small-Angle Neutron Scattering (SANS) Measurements. SANS experiments were performed at the Intense Pulsed Neutron Source at Argonne National Laboratory, Argonne, IL, using the Small-Angle Scattering Instrument (SASI).25 The SASI instrument uses a multiple-aperture collimator consisting of a crossed pair of converging Soller collimators, one for vertical definition and the other for horizontal definition of the angular distribution, with the wavelength resolution ∆λ/λ ranging from 5% to 25%. The β-lactoglobulin/pectin coacervates were transferred to quartz cells 1 mm in thickness. When measuring the solutions of pure β-lactoglobulin, pure pectin, and the mixed solution of β-lactoglobulin/pectin, where D2O is used as solvent to prevent large incoherent scattering from hydrogen, quartz cells with a thickness of 2 mm were employed. All measurements were carried out at 25 ( 1 °C. The data were recorded in the q-range of 0.005-1.0 Å-1 and corrected for detector efficiency, sample transmission, and empty cell scattering. The raw data were analyzed following the procedures described in ref 25. The incoherent background level in the samples was estimated by subtracting the intensity level at a sufficiently large value of q (>0.5 Å-1), which is a constant. Results and Discussion Recently, we have carried out turbidimetric titration26 and dynamic rheology studies27 on the coacervates formed by pectin

Microstructure of β-Lactoglobulin/Pectin Coacervates

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TABLE 1: Composition, Critical Positions of the Shoulder Peak q*, and Average Protein Domain Sizes d for β-Lactoglobulin/ Pectin Coacervates at Various Initial Protein/Polysaccharide Weight Ratios r, Sodium Chloride Concentrations CNaCl, and Pectins with Different Charge Densities coacervates

r

CNaCl (M)

pectin (%)

β-lactoglobulin (%)

H2O%

q* (Å-1)

d (nm)

β-lactoglobulin/pectin A β-lactoglobulin/pectin A β-lactoglobulin/pectin A β-lactoglobulin/pectin A β-lactoglobulin/pectin A β-lactoglobulin/pectin B

10:1 20:1 30:1 30:1 30:1 30:1

0.1 0.1 0.1 0.05 0.2 0.1

1.6 ( 0.2 3.5 ( 0.1 6.5 ( 0.1 1.0 ( 0.2 9.3 ( 0.1 11.7 ( 0.1

18.0 ( 0.1 18.5 ( 0.1 22.3 ( 0.2 18.2 ( 0.1 31.8 ( 0.3 30.6 ( 0.3

80.4 ( 0.6 78.0 ( 0.5 71.2 ( 0.6 80.8 ( 0.8 58.9 ( 0.6 57.7 ( 0.6

0.087 ( 0.001 0.080 ( 0.001 0.093 ( 0.001 0.073 ( 0.001 0.067 ( 0.001

7.2 ( 0.1 7.9 ( 0.1 6.8 ( 0.1 8.6 ( 0.1 9.4 ( 0.2

and β-lactoglobulin. Our turbidimetric titration results revealed that, the pH ranges for the coacervation between β-lactoglobulin and pectin were significantly dependent on the initial protein/ polysaccharide weight ratio (r) and sodium chloride concentration (CNaCl).26 In general, the increase in r promotes the β-lactoglobulin/pectin coacervation. However, the effects of salt are more complicated. Although the addition of salt very often hinders the formation of β-lactoglobulin/pectin coacervates owing to the salt screening effect, β-lactoglobulin/pectin coacervates tend to be more difficult to dissociate at higher salt concentration. In all r and CNaCl studied, β-lactoglobulin/pectin coacervates behave gel-like structures, as evidenced by the much higher storage modulus than loss modulus in our dynamic rheological measurements.27 The elastic properties of coacervates mainly originated from the contribution of pectin chains. β-Lactoglobulin, although mainly contributed to the viscous components, served as the junction points (nodes) of the coacervate network. The plateau modulus (G0) of these coacervate samples is dependent on factors such as r and CNaCl. In the ranges of r up to 40:1 and CNaCl up to 0.2 M, G0 increases monotonously with both r and CNaCl. Because both the phase behavior of protein/polysaccharide coacervation and the physicochemical properties of protein/polysaccharide coacervates are closely related with the structural information of protein/ polysaccharide coacervates, the present work is intended to further investigate the microstructure of β-lactoglobulin/pectin coacervates with the aid of small-angle neutron scattering (SANS). The compositions of β-lactoglobulin/pectin coacervates used in SANS experiments are first analyzed by using the combination of UV, size exclusion chromatography, and dry-weight methods, and the results are listed in Table 1. Although the initial pectin concentration is the same for all β-lactoglobulin/ pectin A coacervates, the pectin A content in the coacervates exhibits an increscent tendency as r increases from 10:1 to 30:1 and as CNaCl increases from 0.05 to 0.2 M. At the same time, the protein amount also shows higher value when these two initial parameters become larger. These results suggest that β-lactoglobulin/pectin A coacervates include larger total amounts of protein and polysaccharide when coacervates are prepared at higher r or higher CNaCl. Because the pectin concentration in the initial mixed solution is the same, higher r will let both protein molecules and pectin chains have more chance to interact to form protein/polysaccharide coacervates. This may be the reason that larger amounts of β-lactoglobulin and pectin A settle into the coacervate phase at higher r. This finding supports Weinbreck et al.’s conclusion that the protein/polysaccharide coacervate is a very flexible system,17 which can adapt to external parameters. The β-lactoglobulin content in β-lactoglobulin/pectin A coacervates is seen to become bigger with increasing r, which is also consistent with whey protein/gum arabic coacervates.17 One unexpected character of β-lactoglobulin/pectin A coacervates is that increasing CNaCl leads to less water content and more protein and polysaccharide amounts in the coacervates. Our protein and polysaccharide composition

analysis results are consistent with our rheological measurements. If we assume that the protein/polysccharide coacervation originates only from the eletrostatic interaction between two oppositely charged components, the added salt should screen the charges of both protein and polysaccharide, thus leading to a more watery coacervate structure. Contrary results on β-lactoglobulin/pectin coacervates suggest that other effects resulting from β-lactoglobulin molecules specifically may play an important role on the coacervate formation. β-Lactoglobulin was reported to aggregate into tetramers, octamers, or even larger aggregates at pH around the isoelectric point.28,29 The stronger tendency of β-lactoglobulin aggregation induced by the higher salt concentration is supposed to repel more water out of β-lactoglobulin/pectin A cocervates. Pectin B carrying lower charges than pectin A will have weaker inhibition on β-lactoglobulin aggregation. The stronger self-aggregation ability of β-lactoglobulin molecules, therefore, is responsible for the observed slightly less watery β-lactoglobulin/pectin B coacervate compared with the β-lactoglobulin/pectin A coacervate at the same sample preparation condition. The above composition analysis results of β-lactoglobulin/pectin coacervates reveal one essential feature of the β-lactoglobulin/pectin system, which is unique because of the self-aggregation character of β-lactoglobulin molecules. To further demonstrate these results, smallangle neutron scattering (SANS) is employed in the following part to delineate the impact of physicochemical parameters such as r, CNaCl, and pectin linear charge density on the microstructure of β-lactoglobulin/pectin coacervates. Effects of Initial Protein/Polysaccharide Weight Ratio. Figure 2 shows the SANS intensities I(q) for β-lactoglobulin/ pectin A coacervates as a function of scattering vector (q) at different initial protein/polysaccharide weight ratios (r) in 0.1 M sodium chloride concentration (CNaCl). These SANS intensity profiles are plotted on a double-logarithmic scale. It is found that all three curves decrease sharply at small q range (0.007 < q < 0.02 Å-1), decrease slightly at intermediate q range (0.04 Å-1 < q < 0.2 Å-1), and eventually converge at large q range (q > 0.2 Å-1). To further understand SANS curves of β-lactoglobulin/pectin coacervates, in Figure 3, we present the SANS intensity profile of mixed solution of 3 wt % β-lactoglobulin and 0.1 wt % pectin A solution at 0.1 M CNaCl with pH 7.0, where there is no binding between protein molecules and pectin chains because of strong electrostatic repulsion between protein molecules and pectin chains.26 For comparison, the SANS curves of pure 3 wt % β-lactoglobulin solution and 0.1 wt % pectin A solution at pH 7.0 are also included in Figure 3. While the pure pectin solution shows a marked upturn at the small q range, there is only a shoulder peak for pure β-lactoglobulin solution at the same q range. Actually, the mixed solution of 3 wt % β-lactoglobulin and 0.1 wt % pectin A solution exhibits the accumulative scattering behavior from pure pectin chains and β-lactoglobulin molecules, including an upturn at the small q range, and a shoulder peak at the intermediate q range. Comparing Figure 2 with Figure 3, the upturn at the small q range for β-lactoglobulin/pectin coacervates is more pronounced

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Figure 2. The small-angle neutron scattering intensity profiles of β-lactoglobulin/pectin A coacervates at various initial protein/polysaccharide weight ratios r in 0.1 M NaCl with pH 4.0.

Figure 3. The small-angle neutron scattering intensity profiles of pure 3 wt % β-lactoglobulin solution, 0.1 wt % pectin A solution, and a mixed solution of 3 wt % β-lactoglobulin and 0.1 wt % pectin A at 0.1 M NaCl with pH 7.0.

than that of the β-lactoglobulin/pectin mixed solution, but both have a similar pattern. Although β-lactoglobulin/pectin coacervates with r ) 20:1 and 30:1 have shoulder peaks at intermediate q range like β-lactoglobulin/pectin mixed solution and pure β-lactoglobulin solution, the peak position is observed to shift to smaller q for β-lactoglobulin/pectin coacervates. The specific characters of SANS curves for β-lactoglobulin/pectin coacervates should be ascribed to the combination of electrostatic interaction of β-lactoglobulin molecules with pectin chains and the self-aggregation of bound β-lactoglobulin molecules on pectin chains. From above, we know that, in the small q limit (q f 0), the SANS intensity profile is mostly dominated by the scattering from the pectin chains

I(qf0) ) FKΒTχT

(1)

where F ) 1/N is the number density (N is the total number of particles) and χT is the isothermal osmotic compressibility of the polysaccharide chains in the coacervate. If the interaction between the polysaccharide chains in the protein/polysaccharide complexes is repulsive, the compressibility will be lowered and the forward scattering will be reduced compared with the case of totally noninteracting polysaccharides. If, on the other hand, the forward scattering intensity is increased, the interaction potential between the polysaccharide chains must be attractive on average, which can lead to more aggregation. Since at pH 7.0 both polysaccharide chains and proteins are negatively charged, the interactions between polysaccharide chains are repulsive and SANS intensity at low q is lower. However, at pH 4.0, the overall charges of proteins are positive. The binding of protein molecules to the pectin chains reduces the repulsive

interaction between pectin chains. Therefore, the scattering intensity at low q is significantly higher for coacervates than that for the noninteracting pectin/β-lactoglobulin mixture at pH 7.0. It is worth noting that the upturns of SANS profiles at small q range for β-lactoglobulin/pectin A coacervates are found to change less pronouncedly with increasing r, and the scattering intensity at small q range has a higher value for β-lactoglobulin/ pectin A coacervates with higher r than those with lower r. When the β-lactoglobulin/pectin A weight ratio increases from 10:1 to 30:1, the change in scattering profile is related to osmotic compressibility of the polysaccharide component in the coacervate. For r ) 30:1, more charges on the polysaccharide chains are screened by a higher amount of bounded protein molecules, making the interactions among pectin chains more attractive compared with the coacervate with r ) 10:1. It is noted that the SANS intensity profiles at intermediate q range show pronouncedly different behaviors for three β-lactoglobulin/pectin A coacervates of different r. The I(q) for r ) 10:1 decreases monotonously with q, whereas a shoulder peak appears in the intermediate q range for β-lactoglobulin/pectin A coacervates at r ) 20:1 and 30:1. In the SANS study on complexes of protein with uncharged30 and charged31 polymers, a correlation peak in the I(q) versus q curve is often observed at similar intermediate q range as our system, which is thought to originate from the long-range electrostatic interactions among charged protein particles. Many investigators18,32 demonstrated that protein/polysaccharide coacervates could be considered as a network of polysaccharide chains generating from the association of protein/polysaccharide complexes, wherein the entangled polysacchride chains are decorated with protein molecules. The association of pectin chains in β-lactoglobulin/ pectin coacervates is responsible for the upturn of the SANS curve at small q range, as discussed above. On the other hand, β-lactoglobulin molecules tend to aggregate with each other.28,29 This specific property of β-lactoglobulin molecules may let us reasonably believe that the protein domains in the present system may be formed from the self-aggregation of β-lactoglobulin molecules, which leads to the appearance of one shoulder peak in the intermediate q range for β-lactoglobulin/pectin A coacervates at r ) 20:1 and 30:1. Usually, the size of protein domains could be estimated from the position of the shoulder peak. The position of the peak at the intermediate q range depends on the spatial arrangement of the proteins and the geometrical structure of each protein molecule.33 Some people used the maximum of the peak to denote the peak position.34 But for the present system, it seems difficult to estimate the peak position from its maximum. Here, we take the critical position (q*) corresponding to the intercept between the slowly varying part of the curve and the linearly decreasing part of the peak position, as shown in Figure 2. From the critical position (q*) of the shoulder peak, we can estimate the average protein domain size (d) using d ) 2π/q*.35,36 The values of q* and d for these two β-lactoglobulin/pectin A coacervates are given in Table 1. In the pH range of 4.0-5.2, β-lactoglobulin dimers will aggregate into oligomeric structure and display a maximum close to the protein isoelectric point.28,29 However, in the more acidic pH lower than 4.0, β-lactoglobulin dimers will dissociate into monomers and there mainly exists an equilibrium between β-lactoglobulin monomers and dimers.28,37,38 At pH 4.0, the aggregation of predominant β-lactoglobulin dimers is affected by many factors such as protein concentration and temperature, as well as salt concentration. When r ) 10:1, the absence of a shoulder peak in the SANS curve suggests the random distribu-

Microstructure of β-Lactoglobulin/Pectin Coacervates

Figure 4. The neutron scattering intensity I(q) from β-lactoglobulin/ pectin A coacervates at different CNaCl with r ) 30:1 with pH 4.0.

tion of the relatively small amount of protein dimers relative to r ) 20:1 and 30:1, and thus no observable protein domains are generated in this β-lactoglobulin/pectin coacervate. At r ) 20: 1, the appearance of a shoulder peak suggests the formation of protein domains among neighboring β-lactoglobulin dimers. The protein domain sizes in β-lactoglobulin/pectin coacervates may not be monodisperse. From the critical position of the shoulder peak q*, the average protein domain size is estimated as 7.2 nm at r ) 20:1. The increase of protein concentration in β-lactoglobulin/pectin coacervates at r ) 30:1 causes the average protein domain size to increase to 7.9 nm. The shoulder peak for β-lactoglobulin/pectin A coacervates at r ) 30:1 is also more pronounced than that at r ) 20:1, which could be a result of the larger amount of electrostatic repulsion between protein domains. Furthermore, a broader shoulder peak in the SANS curve at r ) 30:1 than at r ) 20:1 reflects the more heterogeneous distribution of protein domains within β-lactoglobulin/pectin coacervates at r ) 30:1. Effect of Salt Concentration. Figure 4 shows the SANS results for β-lactoglobulin/pectin A coacervates at different CNaCl with r ) 30:1. All the SANS curves exhibit similar shapes that include an upturn at small q range and a shoulder peak at intermediate q range. Similarly, we can also determine the critical position of shoulder peak q* and calculate the value of average protein domain size d. Table 1 contains the values of q* and d for β-lactoglobulin/pectin A coacervates in three salt concentrations at r ) 30:1. Compared with β-lactoglobulin/pectin A coacervates in 0.05 M CNaCl, the scatterings at small q range in the SANS curve for 0.1 and 0.2 M CNaCl are found to be lower. Higher salt concentration usually leads to looser protein/polysaccharide coacervate structure owing to electrostatic screening between protein molecules and polysaccharide chains.17 Therefore, the observed higher scattering intensity at small q range for lower salt concentrations may suggest tighter association of pectin chains in coacervates, which cause the forward scattering to be higher at low salt concentration. All SANS curves for β-lactoglobulin/pectin A coacervates at these three salt concentrations have a shoulder peak at intermediate q range, suggesting that protein domains are formed in the coacervate network. The protein domain size is 6.8, 7.9, and 8.6 nm for 0.05, 0.1, and 0.2 M CNaCl, respectively. This suggests that higher salt concentration favors the formation of protein domains in β-lactoglobulin/pectin coacervates. Although β-lactoglobulin aggregation decreases with increasing salt concentration at pH close to the isoelectric point of β-lactoglobulin,28 the higher salt concentration will shift the equilibrium between β-lactoglobulin monomers and dimers to the dimeric side at more acidic pH.37,38 Our recent results26 also suggested that the higher salt concentration promotes the self-aggregation of β-lactoglobulin mol-

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Figure 5. The neutron scattering intensity I(q) from β-lactoglobulin/ pectin A coacervate and β-lactoglobulin/pectin B coacervate at 0.1 M NaCl with r ) 30:1 with pH 4.0.

ecules at pH lower than 5.2 owing to the electrostatic screening of protein charges, which is indicated by bigger turbidity values of protein solution at higher salt concentrations. Therefore, a higher amount of salt will not only reduce the binding of protein molecules on polysaccharide chains, but also favor the formation of larger protein domains bound on polysaccharide chains. It is noteworthy that the shoulder peak is more pronounced and broader at 0.1 and 0.2 M CNaCl than at 0.05 M CNaCl, suggesting a more heterogeneous and less-structured coacervate structure with the increase of salt concentration. This observation agrees with the less-ordered coacervates at higher salt concentration in the work of others.17,24 Effect of Pectin Charge Density. The main difference between pectin A and pectin B is the average number of charge groups per disaccharide, or the charge density. Pectin A has 69% galacturonic residues compared with 50% galacturonic residues on pectin B. Figure 5, which gives the SANS intensity profiles for coacervates formed by β-lactoglobulin with pectin A and pectin B, illustrates the effect of pectin charge density on the microstructure of β-lactoglobulin/pectin coacervates. β-Lactoglobulin/pectin A coacervate shows higher scattering intensity than β-lactoglobulin/pectin B coacervate at small q range. This observation may be ascribed to the different charge densities of two pectins. Pectin A carries more negative charges on its chains, which will lead to a relatively larger amount of protein molecules bound on pectin chains. Therefore, protein molecules will screen the charges of pectin A to a larger extent. In β-lactoglobulin/pectin A coacervate, pectin chains carrying protein molecules will then aggregate more tightly to form a network than β-lactoglobulin/pectin B coacervate. From the shoulder peak at intermediate q range, the values of q* and d for these two coacervates can be obtained, as listed in Table 1. The average protein domain size in β-lactoglobulin/pectin B coacervate is 9.4 nm, larger than 7.9 nm in β-lactoglobulin/ pectin A coacervate. Because of the lower charge density of pectin B than pectin A, the interactions between pectin B chains and protein molecules will be somewhat weaker. Consequently, the tendency of formation of protein domains in β-lactoglobulin/ pectin B coacervate is stronger than that in β-lactoglobulin/ pectin A coacervate, leading to the formation of larger protein domains. Microstructure of β-Lactoglobulin/Pectin Coacervates. On the basis of the SANS studies on β-lactoglobulin/pectin coacervates, the schematic pictures of the microstructures of β-lactoglobulin/pectin coacervates are presented in Figure 6. Structure A denotes the microstructure of β-lactoglobulin/pectin A coacervates at r ) 10:1 and CNaCl ) 0.1 M. In this case, β-lactoglobulin dimers are separately bound on the pectin chain network. The disappearance of the protein shoulder peak in the

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Figure 6. Schematic of possible microstructures of β-lactoglobulin/ pectin coacervates.

intermediate q range indicates that there is no observable protein domain formation in the coacervate network. Pectin is one stiff polymer with a persistence length of about 6 nm,39,40 and its binding with protein molecules is relatively weak. This condition may partly give rise to structure B for β-lactoglobulin/pectin A coacervates in other conditions where a shoulder peak exists in the intermediate q range of SANS curves. In these coacervates, neighboring bound proteins can aggregate to form protein domains with average sizes of 6.8-8.6 nm, wherein the electrostatic repulsion between neighboring protein domains produces a clear shoulder peak in the intermediate q range in the SANS intensity profile. The larger amount of protein molecules or higher salt concentration will promote the selfaggregation of β-lactoglobulin molecules to form protein domains with bigger size. At the same physiochemical condition, pectin B with lower charge density helps to form the larger protein domains due to its loose binding with protein molecules, which may also promote self-aggregation of protein molecules. Conclusions In summary, small-angle neutron scattering (SANS) has been used to disclose two kinds of microstructures of β-lactoglobulin/ pectin coacervates formed under different protein/polysaccharide weight ratios, salt concentrations, and pectin charge densities. At CNaCl ) 0.1 M and r ) 10:1, a low protein amount makes protein molecules separately distribute in β-lactoglobulin/pectin coacervate network, and the monotonously decaying SANS intensity profile mainly arises from the scattering from pectin chains. Further increase in r and CNaCl as well as the decrease of pectin linear charge density promote the formation of larger protein domains in the entangled pectin chains, as evidenced by the appearance of a shoulder peak at intermediate q range in the SANS intensity profiles. The formation of larger protein domains is partially due to the high tendency of self-aggregation of β-lactoglobulin molecules. These SANS studies on β-lactoglobulin/pectin coacervates may open up new prospects in understanding the structure of these peculiar complex fluids on a microscopic scale. Acknowledgment. The authors thank Ed Lang at IPNS for his technical support during the SANS measurements. This work was supported by ACS-PRF (41333-G7). References and Notes (1) (2) (3) 23, 1. (4)

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