Article pubs.acs.org/journal/abseba
Fiber-Enforced Hydrogels: Hagfish Slime Stabilized with Biopolymers including κ‑Carrageenan Lukas Böcker, Patrick A. Rühs, Lukas Böni, Peter Fischer,* and Simon Kuster Institute of Food, Nutrition and Health, ETH Zurich, 8092 Zurich, Switzerland ABSTRACT: Hagfish slime, a remarkable soft and elastic hydrogel, is formed by hagfish as a defense mechanism against predation. The extremely fast slime formation, the high water content, and protein threads up to 30 cm in length make it a promising material for the development of hydrogels with embedded fibers. However, under environmental conditions, i.e., in agitation in seawater, the slime collapses. To address the limited structural stability but use the potential of the protein threads as a backbone in fiber enforced materials, we generated composite structures of hagfish slime with biopolymers. Hagfish slime mixed with chitosan reveals that the slime’s mucin fraction has a negative charge due to strong aggregation of both components. The gels formed by κ-carrageenan and starch show synergistic effects by exhibiting high values of water content, elasticity, and viscosity. We demonstrated that in combination with negatively charged biopolymers, fiber enforced hydrogels can be formed. This fiber enforced material has a pronounced cohesiveness and stability, thus combining both properties of biopolymers and hagfish slime. KEYWORDS: hagfish slime, mucin vesicles, protein threads, biopolymers, hydrogel, fiber enforced gel
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INTRODUCTION The slime produced by hagfish is one of nature’s most amazing defense mechanisms. Under attack, hagfish immediately secrete tiny amounts of a glandular exudate. Once in contact with water, the exudate deploys and within seconds forms vast amounts of a fibrous and cohesive slime, which entraps large volumes of water.1 Hagfish exudate contains protein skeins and mucin vesicles (see Figure 1). Exposed to water, both components act synergistically by entrapping large amounts of water. The skeins, in average 150 μm long and 50 μm wide, contain a protein thread composed of intermediate filaments. When fully uncoiled, this thread can reach lengths up to 15−30 cm with a diameter of 1−3 μm.2−8 The protein threads have a high tensile strength and resemble other biomaterials like spider silk.9 The mucin vesicles contain mucin-like glycoproteins.10,11 The membrane of the vesicles is permeable to monovalent ions, but not to polyvalent ions, except for calcium ions, which were found to be necessary for the rupture of the majority of the vesicles of the Pacific hagfish.12,13 Thus, once in contact with water, water diffuses into the mucin vesicles and let them swell. Through convective mixing, the vesicles rupture and release mucin molecules as well as mucin strands (Figure 1). Simultaneously, with the aid of attaching and swelling mucin vesicles, the skeins rapidly uncoil and form a thread network.11,12,14,15 This unique composite structure captures large quantities of water by physically entrapping it. As the water entrapment is transient5 and the mechanical stability of the slime is low, thread aggregation and syneresis of water occur under mechanical stress.16 © 2015 American Chemical Society
Being already a composite system of protein threads and mucins, additional synergistic effects are expected when hagfish slime is mixed with biopolymers. In biopolymer solution, pH value and electrostatic charges are of crucial importance as they determine the colloidal behavior as phase separation, precipitation, or complete miscibility.17 Although hagfish slime is very promising for industrial purposes, the interaction with food grade biopolymers have not been studied yet in detail. By combining hagfish slime with food grade biopolymers, we obtained gels with a high water content and improved rheological properties. ζ-potential measurements are conducted to determine the charges of hagfish slime and food biopolymers to verify the observed phase behavior. By combining the electrostatic and structural information obtained by light microscopy, the flow behavior of the slime−biopolymer mixtures was interpreted. In this study, we showed that by combining hagfish slime with negatively charged biopolymers, a stable mixed gel with integrated threads and increased elasticity and stability was obtained. This is a first approach to incorporate the cohesive and low concentration features of hagfish slime with biopolymer solutions to create new fiberembedded soft materials. Received: September 21, 2015 Accepted: November 10, 2015 Published: November 10, 2015 90
DOI: 10.1021/acsbiomaterials.5b00404 ACS Biomater. Sci. Eng. 2016, 2, 90−95
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Figure 1. Overview of the hagfish slime formation mechanism. Hagfish exudate, which is shown microscopically and schematically on the left, forms hagfish slime in contact with water, shown on the right. Mucin vesicles, shown in green, take up water whereupon mucins, shown in blue, swell until the vesicles break apart releasing mucin and mucin strands. Simultaneously, skeins, shown in red, unfold and mucins bind to the elongated threads.
Figure 2. Concentration dependent rheology of hagfish slime in Milli-Q water. (A, B) Storage and loss moduli as a function of applied strain for hagfish slime at a fixed angular frequency of ω = 1 rad/s. (C) Flow curve from low to high shear rates (full triangles, 0.01 to 100 s−1) and back at high to low shear rates (empty triangles, 100 to 0.01 s−1). (D) Samples of hagfish slime in Milli-Q water after a rheological measurement (left) and hagfish slime lifted up on a spatula (right).
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prefilled with MCT oil (Medium Chain Triglycerides, Delios GmbH, Germany). The approach to stabilize hagfish exudate using oil was first introduced by Ewoldt et al.16 The exudate is slowly emulsified in MCT oil, retaining most efficiently natural composition and functionality of the exudate. The combination of citrate and PIPES in the high osmolarity buffer particularly allows the stabilization of the vesicles. Skeins are stabilized by the buffer but lose their functionality as their unravelling in water is reduced over time. In both cases, stabilized exudate is stored at 4 °C. After exudate sampling, the hagfish were put into fresh seawater for recovery and released afterward. The entire process is in line with the ethical standards approved by Forsøksdyrutvalget (FOTS ID 6912) and was performed under the advice of the Møreforsking Nyhetsarkiv in the Atlanterhavsparken in Ålesund. Import was granted by the Swiss Federal Food Safety and
MATERIALS AND METHODS
Hagfish Exudate Sampling and Stabilization. Hagfish exudate was sampled according to the procedure of Herr et al.12 and Ewoldt et al.16 In brief, Atlantic hagfish (Myxine glutinosa) were caught by fishermen of the Atlanterhavsparken in Ålesund (Norway) in about 100 m depth. The fish were placed in a 10 L bucket of fresh seawater and anesthetized with a 1:9 mixture of clove bud oil (SAFC) to ethanol at a concentration of 1 mL/L. Once unresponsive to touch, the fish were placed on a dissection board and blotted dry. A fish stimulation device (HPG1, Velleman Instruments, 80 Hz, 8−18 V) was used to obtain hagfish exudate. The device electrically stimulates the muscles around the pores, whereby exudate is secreted. Two methods were used to store and stabilize the expelled exudate. Hagfish exudate was either dispersed in stabilization buffer (0.9 M sodium citrate and 0.1 M PIPES at pH 6.7)2,12 or taken up with a pipet 91
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ACS Biomaterials Science & Engineering Veterinary Office (FSVO) and export was granted by Norwegian Seafood Council (Norges sjømatråd). Slime Preparation and Microscopy. Prior to each measurement (rheology, ζ-potential, microscopy), oil-stabilized exudate at a maximum volume ratio of 1:2000 was mixed with 20 mL Milli-Q to form slime. Given the difficult handling of hagfish exudate, the precise amount of hagfish exudate used per slime assay was determined after each measurement by dry weight quantification with a HR73 Halogen Moisture Analyzer (Mettler Toledo, Switzerland). To exclude salt effects on both, the release of mucin from the vesicles and the rheology of the mucin fraction12,13,18 Milli-Q water is used. Another reason is that the final mixtures with biopolymers should be potentially eatable, i.e., low in salt content. The morphology of the slime is studied by light microscopy (Leica DM1000) in bright field mode. Biopolymer Solution Preparation. Hagfish exudate is combined with a variety of typical food-grade biopolymers known for their thickening potential. Solutions of 0.1 and 1 w/w% κ-carrageenan (Danisco, Denmark) and of 1 w/w% starch (commercial corn starch, Unilever, Netherlands) were made by solubilizing the biopolymer in Milli-Q water through heating to 60 °C. Solutions of 1 w/w% chitosan (Molekula, Germany) were prepared by dispersing chitosan at room temperature and partially solubilized in Milli-Q. All solutions were cooled to 10 °C prior to mixing with hagfish slime. Electrophoretic Mobility Measurements. The electrostatic properties (ζ-potential) of the biopolymers were determined with a Zetasizer (Nano Series, Malvern Instruments, Germany) at a concentration of 0.1 w/w% in Milli-Q water. The electrophoretic mobility is measured by Laser Doppler Velocimetry and the ζpotential calculated using the Henry equation and the Smoluchowski μ approximation ζ = ε ε , where η is the viscosity of the medium, μ is the
viscosity. The hysteresis shows that the slime is not stable in shear flow. It is assumed that the network collapses and the water cannot be trapped any longer in the thread-mucin network. It is, however, possible to lift up the entire slime mass by pulling at the thread network showing that threads span across the whole sample (Figure 2D). The results suggest that additional mechanical stability of the slime could create a soft gel with high water content and strong integrity caused by the threads. To address the collapse under mechanical stress and to improve the water holding property of natural hagfish slime, we combined different food grade biopolymers with hagfish slime, namely chitosan, starch, and κ-carrageenan (Figure 3). Slime formation time did not change
0
electrophoretic mobility, ε0 is the permittivity of vacuum, and ε is the dielectric constant of the medium. Besides the polymer solutions, mucin vesicles were analyzed with the Zetasizer in Milli-Q water at a concentration of 0.05 w/w%. Shear and Oscillatory Rheology. Rotational rheometers (MCR501, MCR702, Anton Paar, Austria) equipped with a Couette geometry (CC27, Anton Paar, Austria) were used to study the flow behavior of hagfish slime in Milli-Q water and in the biopolymer solutions. Hagfish slime samples were prepared at different concentrations of 0.01, 0.02, and 0.04 w/w% for oscillatory and shear rheological tests. Frequency sweep experiments at a strain of γ = 1% were applied on the sample followed by amplitude sweeps at an angular frequency of ω = 1 rad/s. The shear viscosity was studied by measuring flow curves recorded at shear rates from 0.01 to 100 s−1 and back from 100 to 0.01 s−1. All measurements were performed at 10 °C to simulate natural conditions.
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RESULTS AND DISCUSSION When hagfish exudate is exposed to water, slime is formed through convective mixing. Within seconds, the mucin vesicles swell and rupture and simultaneously the thread skeins uncoil. Thus, a high water content slime with embedded threads is formed. As shown in a previous study, this material is challenging to measure with a rheometer.16 However, with careful considerations of the geometrical boundary conditions, the physical properties of hagfish slime can be determined through oscillatory and shear rheology (see Figure 2). Hagfish slime is a viscoelastic network with almost equal storage modulus G′ and loss modulus G″ but with a linear viscoelastic regime up to strains of 100% (Figure 2A, B), which is a slightly more extended viscoelastic regime as reported by Ewoldt et al.16 Unique is that these rheological properties arise at dry weight concentrations as low as 0.01 w/w%, which is very close to the reported natural concentration of hagfish slime.5 In shear rate experiments, a hysteresis was observed. As the threads wind up at the measuring geometry (Figure 2C), the slime lost its original network structure and thus its
Figure 3. Hagfish slime combined with (A) chitosan, (B) starch, and (C) κ-carrageenan. Macroscopic (left) and microscopic (right) images of the resulting slime networks.
in the presence of the added biopolymer solutions. After mixing hagfish exudate with an aqueous chitosan solution (1 w/w%), an immediate aggregation and precipitation of hagfish slime and chitosan occurred, leading to a collapse of the structure. Microscopical images show an aggregation of threads, mucin strands, and chitosan particles (Figure 3A). In combination with starch solution (1 w/w%), an opaque phase composed of hagfish slime and starch as well as a watery phase were formed (Figure 3B). This phase separation was also observed microscopically, where stable hagfish slime free of aggregation and a watery phase with only very little hagfish slime components were distinguished. To quantify the physical changes through the addition of hagfish exudate, we rheologically measured the flow properties of this composite 92
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decades in comparison to the pure starch solution (Figure 4B). Moreover, the composite structure was also shear thinning but without the characteristic hysteresis seen for hagfish slime alone (see Figure 2C), indicating an increased mechanical stability. The cohesiveness caused by the threads was still present as the whole solution could be lifted up (see Figure 4C). By combining hagfish slime with starch, a colloidal stable network was formed. In combination with κ-carrageenan, hagfish exudate dispersed homogeneously into the κ-carrageenan solution (Figure 3C). All skeins unravelled to threads, which immobilized and thickened the solution and hence a proper network was formed. Pure 0.1 w/w% κ-carrageenan solution is a viscous liquid without elasticity (Figure 5A) and a low shear viscosity (Figure 5B). Through the combination with hagfish (concentration of 0.05 w/w%), the viscoelastic properties were increased and lead to a pronounced storage modulus G′. The flow curves showed no hysteresis for the pure κ-carrageenan solution but a pronounced hysteresis for the hagfish slime−κcarrageenan mixture. At a 10-fold higher biopolymer concentration (1 w/w%), κ-carrageenan still displayed a viscous dominated response over the entire amplitude range from 1 to 1000% (see Figure 5C). Adding hagfish slime, however, led to a significant increase in both moduli. The storage modulus G′ dominated the rheological response of the mixture and the linear viscoelastic regime lasted up to strains of 100%. The flow curves of pure κ-carrageenan solution and hagfish slime−κcarrageenan mixture showed a shear thinning behavior with higher viscosities for the composite structure (see Figure 5D). In contrast to a lower κ-carrageenan concentration, no hysteresis occurred in shear rheology of the hagfish slime−κcarrageenan mixture. Hence, by adding 1 w/w% κ-carrageenan to the system, hagfish slime was less sensitive to mechanical stress, i.e., lost its disadvantage of a structural collapse. The obtained moduli of hagfish slime with κ-carrageenan do not
structure. Amplitude sweeps of pure starch solution and of the composite structure showed an increase of the storage and loss moduli by two decades (see Figure 4A). In contrast to pure
Figure 4. (A) Amplitude sweeps of 1 w/w% starch solution with and without hagfish slime. (B) Flow curves of 1 w/w% starch solution with and without hagfish slime. (C) Macroscopic image of 1 w/w% starch solution with hagfish slime after rheological measurement.
starch solution (1 w/w%), which showed purely viscous properties, a distinct storage modulus G′ was measurable for strains ranging from 1 to 1000% when hagfish exudate was added. The flow curves from shear rheological measurements showed an increase in the shear viscosity of the mixture by two
Figure 5. Rheological behavior of 0.1 w/w% and 1 w/w% κ-carrageenan solution with and without hagfish slime. (A) Amplitude sweep of 0.1 w/w% κ-carrageenan solution with and without hagfish slime. (B) Flow curve of 0.1 w/w% κ-carrageenan solutions with and without hagfish slime from low to high shear rates and from high to low shear rates. (C) Amplitude sweep of 1 w/w% κ-carrageenan solution with and without hagfish slime. (D) Flow curve of 1 w/w% κ-carrageenan solution with and without hagfish slime from low to high shear rates and from high to low shear rates. (E) Image of hagfish slime in 1 w/w% κ-carrageenan solution taken after rheological measurement. 93
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and existing connection points of the network are multiplied. Second, the spatial arrangement of the threads increased locally the concentration of mucin and the biopolymer, whereby the overall viscosity increased. Repeated oscillatory and shear rheology measurements showed the improved mechanical stability and an improved water holding capacity (as depicted in Figure 5E) in contrast to the natural hagfish slime. In summary, hagfish slime combined with negatively charged biopolymer solutions forms gels with a high cohesiveness, high elasticity, and higher shear viscosity. Furthermore, the combination of hagfish slime with biopolymers corresponds to a combination of structural elements on a macro- and microscale with intermediate composites.
correspond to a simple addition of the individual moduli (hagfish slime plus κ-carrageenan) but indicate a synergistic effect of both components. It has to be mentioned that hagfish exudate contains potassium ions (K+) at 0.1 M,12 which potentially could cross-link κ-carrageenan molecules. Because the 2000-fold dilution yields a K+ concentration below 0.1 mM, the effect can be neglected.19 The colloidal aggregation behavior of the biopolymers and hagfish slime, in particular the difference between starch and κcarrageenan solutions was obtained by measuring the ζpotential of the solutions through electrophoretic mobility tests. Given their large size, hagfish skeins can not be measured in electrophoretic mobility measurements. Thus, we only measured the mucin fraction. The mucin vesicles, stabilized in a buffer solution (0.9 M sodium citrate and 0.1 M PIPES at pH 6.7), are separated from the skeins by centrifugation at 2000 rpm for 2 min. The resulting ζ-potential of the mucin fraction showed a negative value (see Figure 6). Because the protein
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CONCLUSION Natural hagfish slime only persists for a short time (see Figure 7 A). Therefore, we studied the influence of biopolymers on
Figure 6. Zeta potential in mV of mucin in Milli-Q water as well as of 0.1 w/w% polymer solutions of chitosan, starch, and κ-carrageenan. (A) Macroscopic image of the precipitation of mucin in Milli-Q water with chitosan. (B) Microscopic image of the precipitate formed by mucin in Milli-Q water with chitosan.
threads are coated by the mucin fraction,14 it can be concluded that hagfish slime is overall negatively charged. This hypothesis is tested by mixing the isolated mucin fraction in buffer solution with chitosan, which showed a positive ζ-potential. As expected and in line with mucoadhesive properties of chitosan,20 precipitation occurred due to opposite charges of chitosan and mucin (Figure 6A, B). In contrast, mixing the mucin fraction with negatively charged biopolymers such as starch and κ-carrageenan, stable mixtures were obtained as already depicted in Figure 3B, C. The different optical appearance and rheological fingerprint of hagfish slime mixed with starch or κ-carrageenan can be discussed in light of the significant difference in molecular structure and the resulting ζ-potential of both biopolymers (see Figure 6). κ-carrageenan is composed out of a linear chain of galactose and anhydrogalactose unit, both sulfated and nonsulfated and thus provide higher electrostatic interaction than the compact and neutral starch molecule. As a result, the hagfish slime and κ-carrageenan network is stabilized by their strong electrostatic interaction, whereas for starch this effect is smaller. In the composite gels, we assume that the hagfish exudate and the added biopolymer to have a 2-fold effect. First, threads and biopolymer molecules interconnect across the entire slime
Figure 7. Model of the network structure formed by hagfish exudate in water and in differently charged polymer solutions. (A) Exudate in Milli-Q water. The gel is formed in which mucin molecules and mucin strands align to the elongated threads. Upon mechanical stresses the network structure aggregates and collapses. (B) Exudate with a solution of a positively charged polymer. As vesicles rupture and skeins unfold the positively charged polymer simultaneously interacts with the mucins and mucin strands leading to an immediate precipitation. (C) Exudate with a solution of a negatively charged polymer. The gel is formed as in the case of exudate in water. The oppositely charged mucins and polymer molecules repel each other, thus the polymer fills the space between the elongated threads inhibiting the collapse of the system.
hagfish slime stability by electrophoretic mobility and rheological measurements. When positively charged biopolymer chitosan was combined with hagfish slime, the system precipitated as the negatively charged mucin fraction bound to positively charged chitosan. This precipitation demonstrates that positively charged biopolymers are not suitable to act as a sterical barrier against aggregation of hagfish threads, which are coated with negatively charged mucin molecules (see Figure 7B). Negatively charged biopolymers such as starch and κ94
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films from solubilized hagfish slime thread proteins. Biomacromolecules 2012, 13, 3475−3482. (10) Leppi, T. J. Morphochemical analysis of mucous cells in the skin and slime glands of hagfishes. Histochem. Cell Biol. 1968, 15, 68−78. (11) Winegard, T. M.; Fudge, D. S. Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. J. Exp. Biol. 2010, 213, 1235−1240. (12) Herr, J. E.; Winegard, T. M.; O’Donnell, M. J.; Yancey, P. H.; Fudge, D. S. Stabilization and swelling of hagfish slime mucin vesicles. J. Exp. Biol. 2010, 213, 1092−1099. (13) Herr, J. E.; Clifford, A.; Goss, G. G.; Fudge, D. S. Defensive slime formation in Pacific hagfish requires Ca2+ and aquaporin mediated swelling of released mucin vesicles. J. Exp. Biol. 2014, 217, 2288−2296. (14) Koch, E. A.; Spitzer, R. H.; Pithawalla, R. B.; Downing, S. W. Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti). Cell Tissue Res. 1991, 264, 79−86. (15) Luchtel, D.; Martin, A.; Deyrup-Olsen, I. Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish. Tissue Cell 1991, 23, 939−948. (16) Ewoldt, R. H.; Winegard, T. M.; Fudge, D. S. Non-linear viscoelasticity of hagfish slime. Inter. J. Non-Linear Mech. 2011, 46, 627−636. (17) Tolstoguzov, V. B. Functional properties of food proteins and role of protein-polysaccharide interaction. Food Hydrocolloids 1991, 4, 429−468. (18) Bernards, M. A.; Oke, I.; Heyland, A.; Fudge, D. S. Spontaneous unraveling of hagfish slime thread skeins is mediated by a seawatersoluble protein adhesive. J. Exp. Biol. 2014, 217, 1263−1268. (19) Phillips, G. O.; Williams, P. A. Handbook of Hydrocolloids; Elsevier: Amsterdam, 2009. (20) Menchicchi, B.; Fuenzalida, J. P.; Bobbili, K. B.; Hensel, A.; Swamy, M. J.; Goycoolea, F. M. Structure of chitosan determines its interactions with mucin. Biomacromolecules 2014, 15, 3550−3558.
carrageenan, however, provide an optimal barrier against aggregation when embedded into the fiber network (see Figure 7C). Combined with starch, hagfish slime formed a viscoelastic composite structure, which was not significantly stronger than pure hagfish slime but stable against mechanical treatment. With κ-carrageenan, a strong gel featuring a high water content was formed. The hagfish threads were perfectly embedded into the biopolymer network, allowing the gel to recover from stress as the threads did not aggregate at high deformations. The developed technique is simple and allows us to produce fiber enforced gels with an increased elasticity to known hydrogels using only a minimum amount of dry weight. Moreover, the cold set character of the gelled mixtures render the heating and cooling steps necessary for conventional food gels obsolete. Finally, through biomimicry, new fiber enforced gels could be developed that will serve as models for food or pharmaceutical applications.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: peter.fi
[email protected]. Phone: +41 (0)44 632 5349. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We express our thanks to the Aquarium ”Atlanterhavsparken” in Ålesund and in particular to curator Rune Veiseth for kindly providing us hagfish. Møreforsking Nyhetsarki is thanked for the possibility of using their facilities and Snorre Bakke for supervising the sampling of the hagfish according to the ethical guidlines (FOTS ID 6912). Raeffaele Mezzenga is acknowledged for using his Malvern Zetasizer. This work was supported by ETH Research Grant ETH-19 14-1.
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REFERENCES
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