Fibrillation of β-Lactoglobulin at Low pH in the Presence of a

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Fibrillation of β-Lactoglobulin at Low pH in the Presence of a Complexing Anionic Polysaccharide Owen G. Jones, Jozef Adamcik, Stephan Handschin, Sreenath Bolisetty, and Raffaele Mezzenga* ETH Zurich, Food and Soft Materials Laboratory, Institute of Food, Nutrition & Heath, Schmelzbergstrasse 9, LFO E22, 8092 Zurich, Switzerland Received July 2, 2010. Revised Manuscript Received September 3, 2010 The influence of electrostatic complexation with κ-carrageenan was tested on the fibrillation process of β-lactoglobulin at pH 2.0. Morphology and structural development were monitored through cross correlation dynamic light scattering, transmission electron microscopy, and atomic force microscopy. Scattering indicated that noncomplexed β-lactoglobulin monomers aggregated to form fibrils after 15-90 min of heating at 90 °C. However, electrostatic protein-carrageenan complexes found in the unheated system were unchanged by the thermal process. Images and scattering results showed that carrageenan complexes slowed fibrillation kinetics, possibly through reduction in available monomer concentration. Complexes adhered to fibrils at ends and junctions in TEM images, indicating interactive affinity with the fibers, presumably as heterogeneous nucleation sites.

1. Introduction In light of the increasing demand for nutritious and functional foods, the structuring of biopolymer components has become a subject of great interest.1 Biopolymer structures, such as spherical particulates or linear fibrils, can be utilized for the delivery of bioactive components (e.g., vitamins, minerals, or pharmaceuticals)2-4 or for the simulation of desired textures (for instance, whey protein particles as fat mimetics5,6). Fibrillar protein structures may also be used to understand biologically relevant amyloid structures, which are implicated in Alzheimer’s, Parkinson’s, and Creutzfeld-Jakob diseases.7 Using the proper variables, biopolymer structures contribute to both product value and research interests using low-cost, natural components. Many food proteins are known to form fibrils, such as ovalbumin,8 wheat glutenin,9 kidney bean isolate,10 bovine serum albumin,11 soy glycinin,12 R-s2-casein,13 R-lactalbumin,14 and β-lactoglobulin.15 Most likely, this list will be expanded in future years of research as the ability to form fibrils appears to be a *Corresponding author. E-mail: [email protected]. (1) Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Nat. Mater. 2005, 4, 729–740. (2) Chen, L.; Remondetto, G. E.; Subirade, M. Trends Food Sci. Technol. 2006, 17, 272–283. (3) Emerich, D. F.; Thanos, C. G. J. Drug Targeting 2007, 15, 163–183. (4) Ubbink, J.; Krueger, J. Trends Food Sci. Technol. 2006, 17, 244–254. (5) Sandoval-Castilla, O.; Lobato-Calleros, C.; Aguirre-Mandujano, E.; Vernon-Carter, E. J. Int. Dairy J. 2004, 14, 151–159. (6) Janhoj, T.; Ipsen, R. Milchwissenschaft 2006, 61, 131–134. (7) Koo, E. H.; Lansbury, P. T.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9989–9990. (8) Veerman, C.; de Schiffart, G.; Sagis, L. M. C.; van der Linden, E. Int. J. Biol. Macromol. 2003, 33, 121–127. (9) Mackintosh, S. H.; Meade, S. J.; Healy, J. P.; Sutton, K. H.; Larsen, N. G.; Squires, A. M.; Gerrard, J. A. J. Cereal Sci. 2009, 49, 157–162. (10) Zhang, Y.-H.; Tang, C.-H.; Wen, Q.-B.; Yang, X.-Q.; Deng, W.-L. Food Hydrocolloids 2010, 24, 266–274. (11) Veerman, C.; Sagis, L. M. C.; Heck, J.; van der Linden, E. Int. J. Biol. Macromol. 2003, 31, 139–146. (12) Akkermans, C.; van der Goot, A. J.; Venema, P.; Gruppen, H.; Verijken, J. M.; van der Linden, E.; Boom, R. M. J. Agric. Food Chem. 2007, 55, 9877–9882. (13) Thorn, D. C.; Ecroyd, H.; Sunde, M.; Poon, S.; Carver, J. A. Biochemistry 2008, 47, 3926–3936. (14) Goers, J.; Permyakov, S. E.; Permyakov, E. A.; Uversky, V. N.; Fink, A. L. Biochemistry 2002, 41, 12546–12551. (15) Aymard, P.; Nicolai, T.; Durand, D. Macromolecules 1999, 32, 2542–2552.

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highly common property of proteins. However, fibril development has been investigated in only relatively pure solutions, whereas complex media have been largely ignored. Thermal treatment of complex media is common in numerous industries (e.g., food), and the creation or presence of fibrils in treated products should be understood. Interaction between anionic polysaccharides and protein represents a noted complexity among food and pharmaceutical systems. To comprehend fibril development in such a system, β-lactoglobulin fibrillation was investigated in the presence of κ-carrageenan, a polysaccharide with opposing electric charge. β-lactoglobulin is the dominant globular protein found in whey, a soluble extract of milk.16 The native state of β-lactoglobulin, a relatively small protein of 18.4 kDa, is characterized by a β-barrel structure and a neighboring R-helix,17 which, despite differences in surface potential, is nearly identical at pH 7 or 2.18 Inside the central β-barrel lies mostly hydrophobic residues along with six cysteine residues, four of which are involved in disulfide linkages.16 As a lipocalin, the β-barrel is also capable of carrying certain lipophilic chains and may also bear a nutritive function.19,20 Upon denaturation, the β-barrel unfolds, exposing the hydrophobic core.21 Aggregation occurs through hydrophobic agglomeration via the exposed core, hydrogen bonding between β-sheets, and disulfide interchanges. Denaturation (unfolding) and disulfide linkages diminish at acid pH, where the protein is most stable.22 More details of thermally induced β-lactoglobulin molecular changes can be found in the literature.23-27 (16) Sawyer, L. β-Lactoglobulin. In Advanced Dairy Chemistry, 3rd ed.; Fox, P. F., McSweeney, P. L. H., Eds.; Kluwer Academic/Plenum: New York, 2003; Vol. 1, pp 319-386. (17) Brownlow, S.; Cabral, J. H. M.; Cooper, R.; Flower, R.; Yewdall, S. J.; Polikarpov, I.; North, A. C. T.; Sawyer, L. Structure 1997, 5, 481–495. (18) Fogolari, F.; Ragona, L.; Zetta, L.; Romagnoli, S.; de Kruif, K. G.; Molinari, H. FEBS Lett. 1998, 436, 149–154. (19) Wu, S.-Y.; Perez, M. D.; Puyol, P.; Sawyer, L. J. Biol. Chem. 1999, 274, 170–174. (20) Puyol, P.; Perez, M. D.; Peiro, J. M.; Calvo, M. J. Dairy Sci. 1994, 77, 1494– 1502. (21) Qi, X. L.; Holt, C.; McNulty, D.; Clarke, D. T.; Brownlow, S.; Jones, G. R. Biochem. J. 1997, 324, 341–346. (22) Kella, N. K. D.; Kinsella, J. E. Biochem. J. 1988, 255, 113–118. (23) Kontopidis, G.; Holt, C.; Sawyer, L. J. Dairy Sci. 2004, 87, 785–796. (24) Donald, A. M. Soft Matter 2008, 4, 1147–1150.

Published on Web 10/18/2010

DOI: 10.1021/la1026619

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Aggregate structuring of β-lactoglobulin in acidic pH is still a highly debated topic. It has been proposed that aggregation occurs from hydrogen bonding and hydrophobic interactions on the exposed monomer surface. These aggregate structures do not greatly alter the monomer secondary structure but instead cause slight shifts from intramolecular β-sheets to intermolecular β-sheets.28 Kavanagh and others have attributed macromolecular structural differences to electrostatic repulsions alone rather than any effect of thermal unfolding.28 In a series of recent publications, Krebs and others have claimed that amyloid-like crossβ-sheet structuring can be found among all β-lactoglobulin aggregates, regardless of sample pH.29-31 This theory is strengthened by previous FTIR findings where the β-sheet structure shifts were found to be nearly identical at acid and neutral pH values despite small differences in kinetics and peak strength.32 The formation of β-lactoglobulin aggregates thus appears to be mostly dependent on factors such as pH and ionic strength, whereas heating variables mostly affect only the kinetics and yield. A more recent proposal suggests, however, that the fibrils are formed only by hydrolyzed segments of β-lactoglobulin protein arranged in β-sheet secondary structure.33 Despite extensive research on whey protein aggregation, the formation of amyloid-like fibrils from β-lactoglobulin at low pH has been studied for only the last 10 years. It has been proposed that aggregation first occurs among β-lactoglobulin monomers to form dimers, tetramers, or both,34 which then rearrange structurally into antiparallel β-sheets.35,36 These small aggregates would then act as nucleation sites for continued β-sheet interaction that propagates in protein fibrils. The linearity of the fibril chain, noted by a long persistence length, is controlled by strong electrostatic repulsion along the surface of the protein monomers. β-lactoglobulin at low pH is more positively charged and produces fibrils with much longer persistence lengths. Electrostatic repulsions among β-lactoglobulin monomers are also decreased through the addition of ionic strength, which drastically decreases fibril persistence lengths as ionic strength approaches 100 mM.15,37 The major factors influencing the monomer-to-fibril turnover are heating time and concentration. Concentration influences the probability of appropriate monomer interactions. Although a theoretical critical concentration for fibrillation was posted at ∼0.25% (w/w) protein,38 other studies have shown that at least 2% protein (w/w) is necessary to attain a 50% yield.39 The effect (25) Hoffmann, M. A. M.; van Mill, P. J. J. M. J. Agric. Food Chem. 1999, 47, 1898–1905. (26) Hoffman, M. A. M.; van Mill, P. J. J. M. J. Agric. Food Chem. 1997, 45, 2942–2948. (27) Jung, J.-M.; Savin, G.; Pouzot, M.; Schmitt, C.; Mezzenga, R. Biomacromolecules 2008, 9, 2477–2486. (28) Kavanagh, G. M.; Clark, A. H.; Ross-Murphy, S. B. Int. J. Biol. Macromol. 2000, 28, 41–50. (29) Krebs, M. R. H.; Devlin, G. L.; Donald, A. M. Biophys. J. 2009, 96, 5013– 5019. (30) Krebs, M. R. H.; Domike, K. R.; Cannon, D.; Donald, A. M. Faraday Discuss. 2008, 139, 265–274. (31) Krebs, M. R. H.; Domike, K. R.; Donald, A. M. Biochem. Soc. Trans. 2009, 37, 682–686. (32) Lefevre, T.; Subirade, M. Biopolymers 2000, 54, 578–586. (33) Akkermans, C.; Venema, P.; van der Goot, A. J.; Gruppen, H.; Bakx, E. J.; Boom, R. M.; van der Linden, E. Biomacromolecules 2008, 9, 1474–1479. (34) Clark, A. H.; Kavanagh, G. M.; Ross-Murphy, S. B. Food Hydrocolloids 2001, 15, 383–400. (35) Arnaudov, L. N.; de Vries, R.; Ippel, H.; van Mierlo, C. P. M. Biomacromolecules 2003, 4, 1614–1622. (36) Giurleo, J. T.; He, X.; Talaga, D. S. J. Mol. Biol. 2008, 381, 1332–1348. (37) Veerman, C.; Ruis, H.; Sagis, L. M. C.; van der Linden, E. Biomacromolecules 2002, 38, 869–873. (38) Kroes-Nijboer, A.; Venema, P.; Bouman, J.; van der Linden, E. Food Biophys. 2009, 4, 59–63. (39) Schokker, E. P.; Singh, H.; Pinder, D. N.; Creamer, L. K. Int. Dairy J. 2000, 10, 233–240.

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of imparted thermal energy influences aggregation kinetics by increasing monomer unfolding and intermolecular interactions, as indicated by kinetic studies.40 β-lactoglobulin, positively charged below pH 5, is able to form electrostatic complexes with anionic polyelectrolytes and low-molecular-weight anionic surfactants.27 Extensive literature is available on the subject of electrostatic complexation with polyelectrolytes.41,42 In particular, β-lactoglobulin has been shown to form electrostatic complexes with pectins,43 gum arabic,44 acacia gum,45 and κ-carrageenan.46 Complexes with κ-carrageenan are strong due to the highly charged nature of carrageenan and will form coacervates (a biphasic separation) below pH 4.47 Carrageenan is an anionic polysaccharide isolated from seaweed. The negative charge of carrageenan derives from sulfate groups. The three major types of carrageenan, iota, kappa (κ), and lambda, are differentiated by the amount and arrangement of sulfate esters, of which κ-carrageenan possesses the fewest (∼22%).48 The charge density of κ-carrageenan comes to ∼0.92 sulfate ions per disaccharide subunit.49 Carrageenan, because of its high charge, exists as linear rods in simple solution, as loose coils in moderate ionic strength,50 or as intertwining helices among particular cations.51 Carrageenan’s interaction with milk proteins, particularly caseins, is well-established in the stabilization of acidified milk drinks,52 in which the carrageenan acts as both a viscosifying matrix and a contributor of charged elements.53 In the present work, we investigated the fibrillation process of β-lactoglobulin at pH 2 and low ionic strength in the presence of a varying quantity of κ-carrageenan. It was hypothesized that electrostatic interaction between the β-lactoglobulin and an anionic polysaccharide would in some way modify the fibrillation process during thermal treatment. Because of the sulfate groups of κ-carrageenan and its high linear charge density, this polysaccharide was an ideal candidate for strong electrostatic interaction with the protein and is one of the few food-grade biopolymers capable of greatly perturbing the fibrillation process of β-lactoglobulin at a pH as low as 2 (where most other foodgrade biopolymer possesses minimal residual charge). In particular, we will show how coacervation between the protein and the polysaccharide led to a fibrillation process and fiber morphology considerably different from those frequently reported at pH 2 and low ionic strength, and we will discuss the possible mechanisms and main parameters ruling this process. (40) Arnaudov, L. N.; de Vries, R. J. Chem. Phys. 2006, 124, 084701. (41) Cooper, C. L.; Dubin, P. L.; Kayitmazer, A. B.; Turksen, S. Curr. Opin. Colloid Interface Sci. 2005, 10, 52–78. (42) Turgeon, S. L.; Schmitt, C.; Sanchez, C. Curr. Opin. Colloid Interface Sci. 2007, 12, 166–178. (43) Girard, M.; Turgeon, S. L.; Gauthier, S. F. Food Hydrocolloids 2002, 16, 585–591. (44) Weinbreck, F.; de Vries, R.; Schrooyen, P.; de Kruif, C. G. Biomacromolecules 2003, 4, 293–303. (45) Schmitt, C.; Sanchez, C.; Thomas, F.; Hardy, J. Food Hydrocolloids 1999, 13, 483–496. (46) Dickinson, E. Trends Food Sci. Technol. 1998, 9, 347–354. (47) Weinbreck, F.; Nieuwenhuijse, H.; Robijn, G. W.; de Kruif, C. G. J. Agric. Food Chem. 2004, 52, 3550–3555. (48) Campo, V. L.; Kawano, D. F.; da Silva, D. B., Jr.; Carvalho, I. Carbohydr. Polym. 2009, 77, 167–180. (49) Hugerth, A.; Sundelof, L.-O. Biopolymers 2001, 58, 186–194. (50) Croguennoc, P.; Meunier, V.; Durand, D.; Nicolai, T. Macromolecules 2000, 33, 7471–7474. (51) Piculell, L. Gelling Carrageenans. In Food Polysaccharides and Their Applications, 2nd ed.; Stephen, A. M., Phillips, G. O., Williams, P. A., Eds.; CRC Press: Boca Raton, FL, 2006; pp 239-287. (52) Syrbe, A.; Bauer, W. J.; Klostermeyer, H. Int. Dairy J. 1998, 8, 179–193. (53) Alexander, M.; Dalgleish, D. G. Curr. Opin. Colloid Interface Sci. 2007, 12, 179–186.

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2. Experimental Section 2.1. Materials. κ-carrageenan (Fluka BioChemika, lot no. 1432063) was purchased from Sigma-Aldrich (St. Louis, MO). Product solubility was listed at 0.5% (w/v) in hot water, with a viscosity of 15.8 mPa 3 s for a 0.3% aqueous solution. Carrageenan powder was used as received. Hydrochloric acid solutions, for sample acidification, were diluted from a concentrated dispersion (∼37% w/v; Sigma-Aldrich). Pure water was obtained from an on-site filtration unit (NANOpure, 18.2 MΩ 3 cm, Barnstead International, Dubuque, IA). Biopure β-lactoglobulin (lot JE 002-8-415; received Nov 25, 2009) was kindly donated from Davisco Foods International (Le Seur, MN). Reported product specifications were as follows: 98.0% protein (w/w d.b.) with 93.4% β-lactoglobulin (w/w protein), 0.1% fat, 1.0% ash, 4.7% moisture, and pH 6.8 (10% w/v solution). β-Lactoglobulin solution preparation was performed using the procedure of Jung and Mezzenga.54 We created proteins solutions of 10% (w/w) by distributing the powder in pure water, followed by mixing for 2 h using a stir bar and magnetic stir plate (300 rpm, RT). Following solubilization, solutions were adjusted to pH 4.6 using 1.0 N HCl solution and then centrifuged at 15 000 rpm for 15 min at 20 °C (Sigma 3K30H; Rotor 12150-H; 20 400g) to remove precipitating material. Supernatant, containing nonaggregated protein, was removed and adjusted to pH 2.0 using 1.0 N HCl solution. Further removal of aggregates or large impurities was achieved by filtration through a 0.22 μm Durapore filter (GV, 47 mm diameter, Millipor, Billerica, MA), assisted by negative pressure. Ions and sugars were removed from the filtrate by dialysis against (1) 0.01 N HCl solution and (2) pure water using Spectra-Por membranes with a MWCO of 6000-8000 Da (Spectrum Laboratories, Los Angeles, CA). Dialysis membranes were prepared by boiling in 1 mM EDTA solution (pure water) for 10 min, followed by cold storage until use; membranes were rinsed thoroughly with pure water prior to addition of protein solution. Protein solutions were dialyzed for 4 to 12 h periods at 4 °C under constant agitation (magnetic stir plate, 200 rpm) for a total of 5 days, always maintaining a protein solution-to-solvent ratio of 40 (v/v). Following dialysis, protein solutions were adjusted to pH 2.0 using 1.0 N HCl solution and lyophilized. Protein powder was maintained in a dry state until use. 2.2. Solution Preparation. We created solutions of β-lactoglobulin and carrageenan by distributing the carrageenan or lyophilized protein in pure water, followed by mixing using a stir bar and magnetic hot plate (300 rpm). β-Lactoglobulin solutions (1-4 w/w%) were stirred for 2 h at ambient temperature. Solutions of κ-carrageenan (0.01 to 0.2 w/w%) were stirred for 4 h at 40 °C. After full solubilization, solutions were adjusted to pH 2.0 using 1.0 N HCl solution at 21-23 °C. Complexation between β-lactoglobulin and carrageenan was initiated by dropwise addition of carrageenan solution to agitated β-lactoglobulin solution (magnetic stir plate, 300 rpm), both at pH 2.0. To achieve varying carrageenan concentrations, specific volumes of 0.01 N HCl solution (in pure water) were added to fixed volumes of β-lactoglobulin prior to carrageenan addition. All mixtures were stirred (magnetic stir plate, 200 rpm) for 3060 min at room temperature prior to further experimentation or analysis. 2.3. β-Lactoglobulin Fibrillation. Solutions of β-lactoglobulin, with or without κ-carrageenan, were sealed with a magnetic stir bar in heat-durable glass containers (Pyrex) using plastic screw-caps and were placed in a 90 °C oil bath. During thermal treatment, solutions were stirred by a magnetic stir plate, positioned underneath the oil bath, at 250 rpm to avoid localized heating effects. The use of low-shear rates has been shown to increase fibrilization rates for whey protein solutions at pH 2.0 through

Protein and polysaccharide morphologies of unheated and heated samples were imaged using transmission electron microscopy (TEM). Prior to measurement, samples were diluted to 0.2% protein (w/w) using deionized water for improved fibril resolution. Imaging was carried out on carbon-coated copper grids designed for TEM and were glow discharged for 45 s (Emitech K100X, GB) directly prior to sample fixation. Sample grid preparation was as follows: 4 μL of sample dispersion for 1 min, 2 μL of 2% uranyl acetate for 1 s, and 2 μL of 2% uranyl acetate for 15 s. Following each step, the excess moisture was drained along the periphery using a piece of filter paper. Dried grids were examined under vacuum by TEM (FEI, model Morgagni, NL) operated at 100 kV and at magnifications of 10k-20k. All images were selected from at least 20 comparable images. 2.6. Atomic Force Microscopy Measurements. Protein and polysaccharide morphologies of unheated and heated samples, diluted to 0.1% protein (w/w), were investigated in a dry state using atomic force microscopy. A 20 μL aliquot of all solutions was deposited onto freshly cleaved mica, incubated for 2 min, rinsed with Milli-Q water at pH 2, and dried by air. Images were collected using a MultiMode V (Veeco Instruments, Santa Barbara, CA) operated in intermittent mode under ambient conditions. The microscope was covered with an acoustic hood to minimize vibrational noise. Aluminum coated silicon cantilevers (Veeco) with a nominal tip radius of 0.1%), no further additions of κ-carrageenan were made. However, it was apparent, by extrapolation, that the system would have approached neutrality (∼0 mV) if κ-carrageenan concentrations >0.1% were investigated. Pure κ-carrageenan solutions possessed ζ-potential values of -63 mV ( 6 (not shown in Figure 1). (57) Mezzenga, R.; Jung, J.-M.; Adamcik, J. Langmuir 2010, 26, 10401–10405.

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Figure 3. Particle hydrodynamic radius, as determined by dynamic light scattering of β-lactoglobulin, alone or in the presence of κ-carrageenan in pure water at pH 2.

To study the molecular conformation of the individual building blocks prior to the heating process, dried samples of unheated β-lactoglobulin, κ-carrageenan, or both were studied using atomic force microscopy (Figure 2). Images of pure β-lactoglobulin (Figure 2a) show small, spherical particles of approximately 2-5 nm in diameter. Images of κ-carrageenan (Figure 2b) show loose-coil polyelectrolyte structures of approximately 100-200 nm. Mixtures of β-lactoglobulin and κ-carrageenan (Figure 2c) show the development of larger aggregate structures (approximately 25-75 nm) constituted by the electrostatic complexes between the components. Linear segments among the aggregates were resolved in some of the images and attributed to residual loose-coil structures from carrageenan. Numerous small, spherical structures were also evidenced in the image background that bear close resemblance to the pure β-lactoglobulin. Dynamic light scattering was used as a technique to measure the statistical presence of colloidal populations within the unheated samples (Figure 3). Using the CONTIN fit, distinct populations could be resolved among the protein and polysaccharide samples. Solutions of β-lactoglobulin produced a single peak centered at 2 nm, which corresponds very closely to the monomer radius in the literature.17 Mixtures of β-lactoglobulin and κ-carrageenan (top two lines) still showed this monomer/ dimer peak from the lone β-lactoglobulin molecules. However, a second, larger peak, centered at an approximate hydrodynamic radius of 30 nm, had developed. This second peak is attributed to the aggregate particles (possibly complexes) arising in the mixtures, as also found on the AFM image analysis (Figure 2c). Increased carrageenan concentration in the mixture (top line, Figure 3) caused an increase in the relative height of the second Langmuir 2010, 26(22), 17449–17458

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Figure 4. Atomic force microscopy images of (a) β-lactoglobulin alone or in the presence of (b) 0.005 or (c) 0.01% κ-carrageenan after heating at pH 2 and 90 °C for 5 h.

peak with respect to the monomer peak. This suggested that increasing amounts of interacting polysaccharide led to an increase in aggregate concentration without greatly affecting their size. 3.2. Formation of β-Lactoglobulin Fibrils in the Presence of K-Carrageenan. The formation of β-lactoglobulin fibrils at low pH requires a considerable amount of thermal energy. In this experiment, solutions of β-lactoglobulin (2%) were heated for 5 h at 90 °C to produce fibrillar structures at pH 2.0. These structures were evidenced by atomic force microscopy (Figure 4a) and TEM (Figure 5a). Pure protein fibrils possessed persistence lengths up to and beyond the dimensions of the imaging windows for both AFM and TEM because many of the fibrils’ curvatures were minimal. Relatively bright motes on the AFM images (Figure 4a) spaced regularly on the protein structure were indicative of periodicity for a ribbon-like pairing of multiple protein fibrils, as already reported in recent work.58 The effect of adding small quantities of κ-carrageenan to β-lactoglobulin prior to heating was tested to determine the interfering capability of electrostatic complexation. Heated protein-polysaccharide mixtures were investigated by AFM and TEM, which revealed fibrillar structures similar to the protein alone (Figures 4b,c and 5b,c). Among AFM images, the fibrils were largely identical, whereas the background was interspersed with aggregates of 100-500 nm in cross-section (Figure 4b). TEM images (Figures 5b,c) showed similar aggregates, but in addition, their location could be more regularly resolved at the end of fibrils or at a junction between fibrils. This trend provided an insight into the fibrillation mechanism in the presence of counter-charged polyelectrolyte, which will be addressed in the Discussion section. 3.3. Kinetic Development of β-Lactoglobulin Fibrils in the Presence of K-Carrageenan. The growth of fibrils from protein monomer is not instantaneous but rather occurs over the 5 h heating period. To determine if electrostatic complexation with polyelectrolyte affects fibrillation kinetics, β-lactoglobulin with small quantities of κ-carrageenan was heated at 90 °C for increasing lengths of time, quenched to room temperature, and then analyzed for fibril characteristics. Such characteristics include the production of fibrils at the expense of monomers, as determined by dynamic light scattering (Figures 6 and 7) and the detection of short fibril structures using TEM (Figure 8). Samples of pure β-lactoglobulin were run concurrently so as to understand relative differences in structural development fully. (58) Adamcik, J.; Jung, J.-M.; Flakowski, J.; de Los Rios, P.; Dietlier, G.; Mezzenga, R. Nature Nanotechnol. 2010, 5, 423–428.

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The use of cross-correlation dynamic light scattering to detect the starting material (protein monomer), aggregative intermediates (oligomers), electrostatic complexes (complex), and finished product (fibril) offers the unique benefit to study the systems in its real evolving conditions. This methodology allows a statistical description of the entire sample even in the multiple scattering regime, as opposed to single scattering-DLS or to the narrow viewing window offered by imaging techniques. As previously noted, this technique was capable of detecting the presence of protein monomers among unheated mixtures of β-lactoglobulin and/or κ-carrageenan (Figure 3). From these same distributions (bottom lines, Figure 6), the structural development was followed after heating at 90 °C for 15, 30, 45, 90, and 300 min. In the pure β-lactoglobulin sample (Figure 6a), two additional peaks, with respect to the single monomer peak, appeared after only 15 min: peak 2 with center at ∼20 nm (“oligomers”) and peak 3 with center at ∼200 nm (“fibrils”). The peak height of the “fibril” increased with longer heating time. This development came at the expense of the monomer peak, which was nearly negligible after 300 min. The presence of both oligomer and fiber peaks at intermediate times (15 min) suggested that nucleation and growth processes developed simultaneously in the early stages of fibrillation. The introduction of κ-carrageenan to the unheated β-lactoglobulin (Figure 6b) showed very similar characteristics to those of the pure protein, except for the presence of an aggregate peak centered at 30 nm at the onset (0 min). This aggregate peak, previously discussed (Figure 3), resulted from electrostatic complexation between the protein and anionic polysaccharide. Upon heating, the aggregate/complex peak was absorbed into the growing fibril peak at longer heating times (t > 45 min). Greater concentrations of κ-carrageenan (Figure 6c) only increased the size of the aggregate/complex peak in comparison with the β-lactoglobulin monomer peak for all heating times. Absorption of the complex peak into the fibril peak (t > 45 min) indicated that either the fibril signal dominated the scattering pattern at longer heating times or the complexes became physically attached to the fibrils during the process. The latter hypothesis is preferred here, as motivated and discussed later in relation to the TEM images (Figure 8). Three major peak areas could be defined in the dynamic light scattering data: protein monomer (β-lactoglobulin; center=1.5 to 3 nm), electrostatic complexes (center=10-100 nm, with carrageenan only), and fibrils (center = 100-3000 nm). Relative peak areas (%) for these three populations were plotted against heating time to describe the development of fibril structures (Figure 7). In pure protein and β-lactoglobulin/κ-carrageenan DOI: 10.1021/la1026619

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Figure 6. Particle radius as a function of heating time at 90 °C determined by dynamic light scattering of (a) β-lactoglobulin alone or in the presence of (b) 0.005 or (c) 0.01% κ-carrageenan.

Figure 5. Transmission electron microscopy images of (a) β-lactoglobulin alone or in the presence of (b) 0.005% or (c) 0.01% κ-carrageenan after heating at pH 2 and 90 °C for 5 h. Scale bar corresponds to 500 nm.

mixtures, the monomer population decreased steadily with increased heating, demonstrating the aggregation process toward fibrillation. The complex peak (Figure 7b,c) decreased in a similar fashion to the monomer peak, particularly at the longer heating times. The population of fibrils appeared after only 15 min and quickly dominated the distribution. Total peak area of fibrils was maximized after 90 min for all three solutions (data not shown). The gradual disappearance of the monomer and complex peaks and growth of the fibril peaks once again demonstrated the incorporation of the former components into the latter during the thermal process. Kinetic investigations of fibril development were also followed by TEM, with quenched samples taken at 0, 30, 45, and 90 min (Figure 8). Images were also taken at 15 min, but evidence of fibrils or proto-fibrils could not be found in these early stages 17454 DOI: 10.1021/la1026619

(images not shown). Images of β-lactoglobulin show the first evidence of fibrillar development after 30 min of heating, with almost full development at 45 min. Fibrils in the early stages were sparsely populated and surrounded by clusters of protein monomers. The addition of small amounts of κ-carrageenan caused some interesting changes in the apparent structures formed. With 0.005% carrageenan, fibrils were not discovered by TEM after 30 min of heating. Instead, only small aggregate/complex clusters were noted. However, fibrils could clearly be found after 45 min of heating with the κ-carrageenan-β-lactoglobulin complex particles attached or at the ends of the fibrils. Increased carrageenan concentrations (0.01%) produced similar effects on fibril development as lower concentrations, except that the fibrils at 45 min were loosely formed and were composed of clearly resolved protein clusters and complexes. These structures resembled pure protein fibrils at 30 min (Figure 8) and implied an intermediate stage in fibril development accompanied, however, by the presence of electrostatic complexes.

4. Discussion As already anticipated, heating solutions of β-lactoglobulin at low pH induced the formation of protein fibrils through the parallel organization and interaction of β-sheets in the barrel structure. It is also known that at low pH β-lactoglobulin interacts electrostatically with carrageenan, forming complexes and a resulting coacervate phase. These competing interactions for Langmuir 2010, 26(22), 17449–17458

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Figure 7. Population of discrete size distributions for (a) 0.1% β-lactoglobulin solution alone or in the presence of (b) 0.005 or (c) 0.01% κ-carrageenan as a function of heating time, determined via cross correlation dynamic light scattering.

β-lactoglobulin at low pH present unique possibilities in both structural formation and kinetic development. To solve this dilemma, we discuss in detail the morphology and kinetics of complex formation between β-lactoglobulin and κ-carrageenan at pH 2.0 before and after heat-treatment and their impact on the fibrillation process. 4.1. Characterization of β-Lactoglobulin and K-Carrageenan at pH 2.0. Titration of pure β-lactoglobulin with κ-carrageenan showed an initial increase in charge (ζ potential) before decreasing toward neutrality (Figure 1). Many proteins are known to be positively charged at acidic pH because of the ionization of basic amino acids and the protonation of acidic amino acids. At pH 2.0, β-lactoglobulin is below its isoelectric point (pI ∼5.1) and possesses a positive-to-negative charge ratio of ∼1000.16 κ-Carrageenan possesses a strong negative charge at pH 2.0 due to sulfate functional groups; these interact electrostatically with the protein when mixed in solution.47 The addition of small carrageenan concentrations to the pure protein induced insignificant adjustments to charge until reaching 0.02%, where charge reductions were noted. Average protein charge is only diminished when a significant molar fraction of the protein is complexed with negatively charged carrageenan. By studying mixtures with carrageenan concentrations below 0.02%, we were able to investigate fibrillation of protein in the presence of complexes, as opposed to studying thermal aggregation between Langmuir 2010, 26(22), 17449–17458

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complexes, which possess minimal surface charge. Higher carrageenan concentrations (>0.1%) exhibited excessive interaction and precipitation in the unheated system, making interpretation of the fibrillation process unreliable. Atomic force microscopy of unheated β-lactoglobulin or κ-carrageenan solutions demonstrated the presence of individual, unaggregated protein monomers, and loose-coil polyelectrolytes (Figure 2a,b). Lone β-lactoglobulin monomers are known to exist at pH 2 because of high surface charge, which is made possible at reduced ionic strengths.18 The unheated κ-carrageenan images agree with AFM images of κ-carrageenan in the absence of added salts, showing a distribution of modestly linear coils.59 Mixtures of β-lactoglobulin and κ-carrageenan (Figure 2c) revealed the structural morphology of an electrostatic complex formed between the positively charged protein and negatively charged polysaccharide. As shown, it was possible to resolve numerous protein spheres complexed along the carrageenan coils (note small protuberances sporadically placed along the coil). After extended incubation times (room temperature), these coil-like structures disappeared in favor of larger spherical aggregates (also seen in the periphery of Figure 2c). Excess protein monomers in the background demonstrated the inequality between positively charged protein and negatively charged polysaccharides. This was further reinforced by dynamic light scattering, as peaks attributed to the β-lactoglobulin monomers were evidenced in β-lactoglobulin/carrageenan mixtures (Figure 3). The complexes formed, despite possessing a much larger size (detected diameter ∼30 nm), were not in significant presence to overshadow the monomer signal (∼2 nm). Because of the large excess of protein in the system, it is highly probable that the carrageenan coils were heavily coated with protein, as the positively charged monomers sought to interact with the limited quantity of negatively charged polysaccharide sites. It is postulated that the complex surface was dominated by protein. 4.2. Formation of β-Lactoglobulin Fibrils in the Presence of K-Carrageenan. Solutions of pure β-lactoglobulin at pH 2.0 after thermal treatment (90 °C, 5 h) demonstrated high concentrations of fully developed protein fibrils (Figures 4a and 5a). Evidence of periodicity was found in the AFM images (noted from relative bright and dark spots along the fiber), which have been previously found for β-lactoglobulin fibrils at pH 2.0 at low ionic strengths. Periodicity, indicative of twisting fiber quaternary structuring, occurs from strong positive-charge repulsions between the protein components at low pH.58 Twisting structures minimize surface-to-surface contact while maintaining hydrophobic and hydrogen bond linkages thought to be responsible for interfibrillar coordination. High persistence lengths have been similarly attributed to electrostatic repulsions among proximal protein components. When κ-carrageenan was electrostatically complexed with some of the β-lactoglobulin, a complex/aggregate structure was shown to develop (Figures 2 and 3). Following thermal treatment, fibrils of similar characteristic were seen to form regardless of whether complexes were in solution, as studied by AFM and TEM (Figures 4b and 5b,c). These images also revealed that the complex structures were still present with very little change in their morphology. The presence of electrostatic complexes prior to heating could lead to three major hypothetical changes in the thermal-induced fibrillation process: (1) the electrostatic complex dissolves, leaving the protein to form fibrils, which interact with carrageenan again (59) Funami, T.; Hiroe, M.; Noda, S.; Asai, I.; Ikeda, S.; Nishinari, K. Food Hydrocolloids 2007, 21, 617–629.

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Figure 8. Transmission electron microscopy images of pure β-lactoglobulin alone or in the presence of 0.005 or 0.01% κ-carrageenan as a function of heating time at 90 °C. Scale bar corresponds to 500 nm.

after cooling; (2) only free protein forms fibrils, whereas complexed protein does not participate in fibrillation; and (3) complexed protein maintains its interaction with carrageenan, but exposed protein surfaces act as reactive sites for free protein interaction and participate in the fibrillation process. Electrostatic complexes between β-lactoglobulin and carrageenan should theoretically not dissolve upon heating because electrostatic interactions are not sufficiently weakened in the temperature regimes studied.60-62 This stands in contrast with heating complexes with weakly charged polysaccharides (such as pectin), which are more reliant on hydrogen bonds; these complexes partially dissociate under mild heat treatment.43,63 Also, images revealed that complex structures remained after the entire heat treatment procedure (Figures 4 and 5). The location of these aggregates was shown to adhere always to the fibers (AFM), or, more interestingly, to mostly occur at the ends of fibers (TEM). These facts taken together would suggest that the fibers nucleate at the surface of (60) Pink, D. A.; Hanna, C. B.; Quinn, B. E.; Levadny, V.; Ryan, G. L.; Filion, L.; Paulson, A. T. Food Res. Int. 2006, 39, 1031–1045. (61) Hammond, M. R.; Mezzenga, R. Soft Matter 2008, 4, 952–961. (62) Kaibara, K.; Okazaki, T.; Bohidar, H. B.; Dubin, P. L. Biomacromolecules 2000, 1, 100–107. (63) Jones, O. G.; Decker, E. A.; McClements, D. J. Food Hydrocolloids 2010, 24, 239–248.

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β-lactoglobulin-κ-carrageenan complexes and propagate away from them (hypothesis 3), although it is equally probable that complexes interacted with the formed fibrils after heating (hypothesis 2). 4.3. Kinetic Development of β-Lactoglobulin Fibrils in the Presence of K-Carrageenan. The use of cross-correlation dynamic light scattering allowed us to track the monomer population, even in the presence of large fibrils and electrostatic complexes, and up to the latest stages of aggregation (Figures 6 and 7). TEM was used as a complementary technique to probe the structure of the fibers and aggregates at the molecular level (Figure 8). DLS revealed populations of monomers (1.5 to 3 nm), oligomers (10-30 nm), electrostatic complexes (10-100 nm), and fibrils (100-1000 nm). For the pure protein solutions, a peak between 10 and 30 nm was attributed to the formation of oligomers. These oligomers were likely precursors of fibrils (proto-fibrils) or more generally intermediate aggregate formations because these peaks disappeared at longer heating times. In protein solutions containing carrageenan, early aggregate species of the protein (oligomers) could not be distinguished from the electrostatic complexes because their peaks overlapped. We could clearly follow the formation of fibrils at the expense of monomers by studying the peak area development in DLS Langmuir 2010, 26(22), 17449–17458

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analysis (Figure 7). These curves show the gradual loss in β-lactoglobulin monomers and sudden increase in fibrils over the heating regime. Fibril peaks were diminished in samples containing carrageenan between 15 and 45 min. Also, TEM images indicated evident production of fibrils after 30 min in the pure protein system, whereas fibrils were only observable after 45 min among samples with carrageenan (Figure 8). These observances showed that relative concentration of added carrageenan contributed a weakening effect on fibril formation. Systems containing greater carrageenan concentrations (0.01%) possessed weaker “fibril” peak intensities at 30 and 45 min compared with lower concentrations (0.005%) (Figure 7b,c). Images of fibrils also demonstrated more poorly formed fibrillar aggregates in solutions containing 0.01% carrageenan at 45 min (Figure 8). Therefore, either the anionic polysaccharide (κ-carrageenan) or the resultant electrostatic complex with β-lactolgobulin reduced fibrillation kinetics during thermal treatment. Reduction in fibrillation kinetics with carrageenan could be explained by three different scenarios: (i) complexation reduces the concentration of available β-lactoglobulin monomers, curtailing the fibrillation kinetics; (ii) κ-carrageenan, as a polyelectrolyte, increases the effective ionic strength, which reduces fibril formation; and (iii) κ-carrageenan interacts at specific locations along the β-lactoglobulin monomer, blocking fibril formation. In strong support of (i), protein concentration is known to produce a strong influence on fibrillation kinetics; fibrillation of proteins below 2% (w/w) protein concentration has been shown to be drastically diminished.35,39 Alternatively (ii), the presence of charged carrageenan molecules could contribute to the ionic strength of the solution, which has negative influence on fibrillation. For example, it is known that >80 mM NaCl will significantly decrease persistence lengths of whey protein fibrils.15,37 However, given that κ-carrageenan has an approximate charge of 0.92 per disaccharide subunit, the ionic strength due to sulfate groups is well below 80 mM, and electrostatic screening remains unlikely. Finally (iii), it is possible that κ-carrageenan interacts with β-lactoglobulin residues critical to fibrillation. β-Lactoglobulin, like many relevant proteins, possesses short polypeptides (termed minimal sequences) that are believed to form intermolecular β-sheet alignments.64 After alignment, these minimal sequences theoretically act as templates for further aggregation. Although the identity of such a minimal sequence on β-lactoglobulin still appears inconclusive, it is conceivable that κ-carrageenan interacts with such a polypeptide and interferes in the β-sheet alignment necessary for fibrillation. Nonetheless, relevant ionic strength was too low (ii), and it remains difficult to prove interaction with minimal sequences (iii); therefore, reduced protein concentration is the likely parameter responsible for the different kinetics of fibrillation (i). Peaks assigned to electrostatic complexes (10-100 nm) possessed greater area with increasing carrageenan concentration, as would be expected for the introduction of an interactive species. Most, if not all, carrageenan was expected to interact electrostatically with β-lactoglobulin monomers, yet the relative composition of complexes in solution would remain low because of the large difference in protein and polysaccharide concentration. Quite consistently, TEM images indicated that these electrostatic complexes persisted and tended to be found in agglomerations with fibrils at all times (Figure 8). This persistence indicates that the electrostatic complex did not dissolve during thermal treatment, as expected by simple energetic arguments.61 Relative peak (64) Euston, S. R.; Ur-Rehman, S.; Costello, G. Food Hydrocolloids 2007, 21, 1081–1091.

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areas attributed to these complexes, however, diminished with prolonged heating (Figures 6 and 7). After sufficient thermal exposure (90-300 min), these peaks merged with the growing fibril peaks, creating a large peak width between 10 and 3000 nm. Combined, these results further reinforced hypothesis 3 (Section 3.2) that electrostatic complexes of β-lactoglobulin and κ-carrageenan do not dissolve but instead act as a nucleation site for β-lactoglobulin fibrillation. The presence of the proposed electrostatic complexes at the end or at junctions of fibrils (Figures 5 and 8) led us to strongly support the protein on the surface of complexes as nucleation sites for protein fibrillation during heating (hypothesis 3). Electrostatic complexes, because of their increased composite mass, possess reduced Brownian motion and could present a desirable nucleation site for protein aggregation among heated solutions. The formation of amyloid fibrils has been attributed to the misfolding of only a minor fraction of protein; this misfolded protein then acts as a template (or nucleation site) for further β-sheet alignment.65 β-Lactoglobulin, complexed with κ-carrageenan, may have restructured and acted as such a template. Similarly, fibril initiation has been linked with protein aggregation,36 particularly among partially unfolded β-lactoglobulin monomers;66 the electrostatic complex, as a specific type of aggregate, may serve as such a nucleation site. It is worth stressing further that hypothesis 2, which argues interaction among fibers and complexes after fiber growth (e.g., adhesion versus heterogeneous nucleation), cannot be completely ruled out in this stage. Hypothesis 2 can be similarly used to explain the trends among DLS diagrams of the complexes and growing fibers peaks. Ongoing efforts are devoted to elucidating this point further.

5. Conclusions The formation of fibrils from heat treatment of β-lactoglobulin is influenced by a variety of factors, including temperature, heating duration, protein concentration, and ionic strength. In the present work, we have studied the complexation of the protein building blocks with κ-carrageenan as a new possible factor to affect the aggregation and fibrillation schemes. Electrostatic complexes were shown to form between κ-carrageenan and β-lactoglobulin through a reduction in protein ζ potential and by images of spheroid aggregates already in unheated systems. Exposing these mixtures to high temperature induced the formation of long, linear protein fibrils. Fibril formation was delayed by presence of complexes, but fibril characteristics were unaffected by their presence. Complexes were also unaffected by the thermal process and were often found at the ends or at junctions between fibrils. To ascertain the role of the complex in the formation of protein fibrils, more extensive investigations in the fibrillation process were pursued. Kinetics of fibrillation, followed by dynamic light scattering and TEM, was able to show the development of fibrillar structures at the expense of protein monomers within the first 90 min. Complexes were shown to persist during thermal treatment, but the scattering signal merged with the fibril into a signal scattering object in later heating stages. Electrostatic complexes appeared to delay the appearance of formed fibrils (from 30 to 45 min), as indicated by extensive imaging of samples by TEM. Ultimately, the fully formed fibrils after 300 min of heating were indistinguishable, regardless of the presence of κ-carrageenan, although (65) Malolepsza, E.; Boniecki, M.; Kolinski, A.; Piela, L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7835–7840. (66) Hamada, D.; Dobson, C. M. Protein Sci. 2002, 11, 2417–2426.

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the electrostatic complexes were still evident. The presence of the complex at ends or junctions of the formed fibrils as well as merged peaks in DLS suggested that complexes possessed binding affinity for the formed fibrils or, more likely, that proteins on complex surfaces acted as heterogeneous nucleation sites for fibrillation.

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Results from this work could be applied toward future understanding of the fibrillation process. In particular, modulation of fibril formation needs to be understood in the presence of interactive or noninteractive polymeric materials, because these represent a significant contribution to complex media within the food and pharmaceutical industry.

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