Mapping the Complex Phase Behaviors of Aqueous Mixtures of Îº

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Mapping the Complex Phase Behaviors of Aqueous Mixtures of #-Carrageenan and Type B Gelatin Yiping Cao, Lu Wang, Ke Zhang, Yapeng Fang, Katsuyoshi Nishinari, and Glyn O. Phillips J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b05002 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 11, 2015

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The Journal of Physical Chemistry

Mapping the Complex Phase Behaviors of Aqueous Mixtures of κ-carrageenan and Type B Gelatin Yiping Cao,a Lu Wang,a Ke Zhang,a, b Yapeng Fang,*, a, b Katsuyoshi Nishinari,a, b and Glyn O. Phillips a a

Glyn O. Phillips Hydrocolloid Research Centre, School of Food and Pharmaceutical Engineering,

Faculty of Light Industry, Hubei University of Technology, Wuhan 430068, China b

Hubei Collaborative Innovation Centre for Industrial Fermentation, Hubei University of

Technology, Wuhan 430068, China *

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ABSTRACT: We report a detailed and complete phase diagram for an aqueous mixture of oppositely charged gelling biopolymers, type B gelatin and κ-carrageenan (KC) at pH 7.0. The phase diagram is studied in the ionic strength-temperature coordinate by means of turbidity, rheological and differential scanning calorimetric measurements as well as by macroscopic phase compositional analysis. Seven phase regions are identified, including: I) compatible region; II) electrostatically induced associative phase separation (EIAPS) region; III) hydrogen bonding induced associative phase separation (HBIAPS) region; IV) coexistence of EIAPS and HBIAPS; V) segregative phase separation (SPS) region; VI) coexistence of HBIAPS and SPS; VII) SPS trapped by gelation. The HBIAPS reported for the first time here is attributed to the extensive hydrogen bonding formation between gelatin and KC above their conformational transition temperatures, as probed by addition of urea and methylene blue as well as by 2D 1H-1H NOESY NMR. NaCl is found to have dual effects on HBIAPS. The electrostatic complexation at lower ionic strength facilitates the formation of hydrogen bonds between gelatin and KC, and hence the HBIAPS. It is believed that the local structural arrangement of gelatin molecules or the change in local solvent environment prior to triple helix formation during cooling enables the formation of hydrogen bonds with KC.

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INTRODUCTION Phase separations of biopolymer mixtures are useful in structuring soft materials for a wide range of applications in the food, cosmetic and pharmaceutical industries. Careful design of the final morphology of phase separating systems can be used to create new and desired properties that individual biopolymers fail to provide.1-4 The phase separation of two biopolymers in a common solvent may be divided into associative and segregative phase separation.5, 6 An effective repulsion between biopolymers or an asymmetry in biopolymer-solvent interaction may result in a segregative phase separation in which each phase is enriched in one of the biopolymer components. Associative phase separation is characterized by a phase enriched in both components, in equilibrium with a dilute phase containing mostly solvent, as a consequence of an effective attraction between biopolymers or strong biopolymer-solvent repulsions. The coexistence of associative and segregative phase separations is possible under certain circumstances, which is a thermodynamically non-equilibrium state or kinetically trapped by gelation.7, 8 According to Piculell5 and Doublier,6 both associative and segregative phase separation can be explained by the Flory-Huggins theory in the framework of biopolymer-biopolymer-solvent interactions of a ternary system. The segregative phase separation occurs when the Flory-Huggins interaction parameter (characterizing the biopolymer-biopolymer interaction) is positive and a net repulsion exists between the biopolymers. Electrostatic repulsion between similarly charged biopolymers or strong solvent-biopolymer interaction would elicit the demixing of the biopolymers. The associative phase separation occurs when the Flory-Huggins interaction parameter is negative and the interaction between biopolymers is attractive. This is the case when the biopolymers carry opposite charges. In principle, hydrogen bonding or hydrophobic interaction between biopolymers

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could also induce associative phase separation, although it was barely reported. Associative phase separation of a ternary mixture is often referred to as complex coacervation.6, 9 Parameters influencing biopolymer-biopolymer and biopolymer-solvent interactions, such as biopolymer type, molecular weight, flexibility, hydrophilicity/hydrophobicity, charge density, pH, ionic strength, and temperature etc., have impact on the type and extent of the biopolymer phase separations.4, 10, 11 It is possible to tune the phase separation behaviors of biopolymer mixtures and their resulting morphologies and properties by controlling the relevant parameters. pH, ionic strength and temperature are the most exploited. Additionally, they are also important factors controlling the conformational ordering and gelation of biopolymers. This leads to complicated yet interesting interplays between phase separations and conformational ordering/gelation.12-14 It has been reported that conformational ordering in a biopolymer mixture can lead to segregative phase separation,7,

8, 15, 16

which is attributed to: 1) an increased molecular mass during

conformational ordering, leading to an increased thermodynamic incompatibility between the biopolymers; 2) an increased asymmetry in biopolymer-solvent interaction upon conformational ordering, leading to a reduction in biopolymer-biopolymer interaction. Moreover, gelation or viscoelasticity effect often accompanies conformational ordering. These effects are known to generate dynamic asymmetry between the two biopolymer components of the mixture, and if coupled to phase separation, would lead to a complicated situation called as “viscoelastic phase separation”.14, 16, 17 For example, gelation can retard phase separation to different extents and/or arrest phase separations at certain stages, resulting in completely different microstructures and mechanical properties of the biopolymer mixture.14, 16, 18, 19 More general dynamic asymmetry during viscoelastic phase separation can result in much richer microstructures and morphologies for biopolymer mixture.14, 16 4


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κ-Carrageenan (KC), a negatively charged polysaccharide extracted from marine red algae, is composed of alternating α-(1-3)-D-galactose-4-sulfate and β-(1-4)-3,6-anhydro-D-galactose repeating units.20-22 KC is widely used as a thickener, gelling agent, and stabilizer in the food industry.8 It undergoes a conformational transition from random coil to double helix during cooling, leading to the formation of elastic gels.20 The conformational ordering of KC is largely influenced by salt type and concentration.20, 23 Gelatin is a denaturated product from collagen through a hydrolysis process. The native collagen molecule is a right-handed super triple helix formed by three individual molecular strands held together by interchain hydrogen bonding.24, 25 Gelatin is a polyampholyte molecule whose net charge depends on pH. Upon cooling, the denatured gelatin molecule could partially revert into the original triple helical conformation, resulting in the formation of a gel structure.24, 26 It was found in previous studies that the mixture of KC and type B gelatin exhibited different phase behaviors, including compatible, segregative or associative phase separations7, 8 A coexistence of segregative and associative phase separations was observed and it was attributed to a kinetically trapped state by gelation. The present work revisits thoroughly the complex phase behaviors of the mixture, based on turbidity, DSC and rheological measurements as well as by phase compositional analysis. This leads to a detailed and complete phase diagram and also for the first time the identification of a hydrogen bonding induced associative phase separation (HBIAPS). Mechanistic studies on the HBIAPS are carried out using urea and methylene blue as probes and by 2D 1H-1H NOESY NMR.

MATERIALS AND METHODS Materials. Type B bovine bone gelatin (batch no. G9382) was purchased from Sigma Co. Ltd. Its weight average molar mass (Mw = 173 kDa; Mw/Mn = 2.0) and radius of gyration (Rg = 25.9 nm) 5



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were determined by gel permeation chromatography-multiangle laser light scattering (GPC-MALLS) (Wyatt Technology Corporation, USA) using phosphate buffer (1/15 M, pH 7.0) as eluent. The temperature for GPC-MALLS was 25 oC, which was slightly above the conformational ordering temperature (22 oC) of the gelatin at this salt concentration. The measured molecular parameters therefore represent mostly the random coil conformation of gelatin, but could also include partial contribution from aggregated forms of single chains.27 The isoelectric point of the gelatin was determined to be IEP = 5.1 by Nano-ZS ZetaSizer (Malvern Instruments, UK). KC was obtained from FMC biopolymer (Gelcarin GP-911 NF). It was purified by extensive dialysis against Milli-Q water, followed by conversion into sodium type using ion exchange resin (Amberlite IR-120, Sigma). The freeze dried sample was used for all the experiments. Its ionic composition (wt%) determined by atomic absorption was: 6.32% Na; 0.067% K; 0.0027% Mg; 0.0083% Ca. GPC-MALLS measurement for KC was conducted at 25 oC using 0.1 M NaI as eluent, which ensured a double helical conformation of KC without helical aggregation.28, 29 The measured molecular parameters were: Mw = 467 kDa; Mw/Mn = 1.2; Rg = 85.0 nm. All the other chemical reagents used in the study were of analytical grade. Sample Preparation. Stock solutions of gelatin and KC at 1.50 wt% were prepared by dissolving appropriate amounts of the samples in NaCl solutions of various concentrations. The dispersions were heated for 1 hour at 60 °C and 85 °C for gelatin and KC, respectively, under magnetic stirring. The dissolving temperatures were chosen to minimize the possible thermal degradation of gelatin and KC. Mixtures of 0.75%KC/0.75%gelatin were prepared by mixing equal amounts of the stock solutions, followed by stirring at 85 °C for 10 mins. The pH of the mixtures were checked and adjusted to pH = 7.0 using 1 M NaOH or HCl. Turbidity Measurements. Turbidity change as a function of temperature during cooling for 6


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KC/gelatin aqueous mixtures was measured on a TU-1900 UV/Vis spectrophotometer (Persee, China). The samples were placed in a copper cuvette fixed with optical quartz windows. The temperature was controlled by a Peltier device (Quantum Northwest, USA) at a cooling rate of 0.5 °C/min. Turbidity at 500 nm was calculated as follows:

τ = ( − 1 L) ln( I 0 I t )

Where L is the optical path length (1 cm), Io the incident light intensity, and It the transmitted light intensity. Differential Scanning Calorimetry (DSC). DSC measurements were conducted on a high-sensitivity microcalorimeter DSC-III (Setaram, France). About 0.7 g of sample was hermetically sealed into a stainless cell and an equal amount of NaCl solutions was used as reference. The sample was heated from room temperature to 70 °C at a scan rate of 3.0 °C/min and was held at 70 °C for 10 min. The subsequent cooling process from 70 °C to 0 °C at a scan rate of 0.5 °C/min was recorded. Rheological Measurements. Temperature dependence of storage (G') and loss (G'') moduli of KC/gelatin aqueous mixtures upon cooling was measured on a HAAKE RheoStress 6000 rheometer (Thermo Fisher Scientific, USA), using dynamic oscillation mode at a frequency of 1 Hz and a stress of 0.5 Pa. The stress was checked to be within the linear viscoelastic region. A serrated parallel-plate geometry (diameter 35 mm; gap 1.0 mm) was used to prevent slippage. Samples were loaded onto the geometry pre-heated at 70 °C and were covered with a thin layer of silicon oil to prevent evaporation. The temperature was controlled by an external circulating water bath from 70 °C to 10 °C at a cooling rate of 0.5 °C/min. Dynamic Light Scattering. Change in particle size as a function of temperature for KC/gelatin

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aqueous mixtures was measured using dynamic light scattering on a Nano-ZS Zetasizer apparatus (Malvern Instruments, U.K.). The apparatus was equipped with a 10 mW He−Ne laser emitting at 633 nm. The intensity autocorrelation function was recorded using backscattering mode at an angle of 173o, and was analyzed by the second-order cumulant fit to determine z-averaged diffusion coefficients Dz.30,

31

Particle size, characterized by z-averaged hydrodynamic diameter Dh, was

calculated from Dz according to the Stokes−Einstein equation:30, 31 ‫= ݖܦ‬

݇‫ܶ ܤ‬ 3ߨߟ‫ܦ‬ℎ

where η is the medium viscosity and kBT is the thermal energy. Methylene Blue Spectrophotometry. A methylene blue (MB) spectrophotmetric method was used to probe the interaction between KC and gelatin, as previously described by Michon et al.32 The method is based on the principle that the planar cationic dye can interact with the regularly spaced sulphate groups of KC to form long-range metachromatic complexes. The metachromatic complexes require a close and parallel arrangement of MB molecules (i.e., 0.35-0.77 nm) to absorb at 554 nm. This absorption is distinct from that of 663 nm from free MB molecules. Interaction of KC with gelatin would modify the absorption spectrum of KC/methylene blue. MB at a final concentration of 0.001wt% was added to 0.005% KC, 0.75% gelatin or their mixture. The solutions were stirred at 85 °C for 10 min, and then loaded on to a TU-1900 UV/Vis spectrophotometer (Persee, China) for absorption measurements. Distilled water was used as a reference. Absorption as a function of wavelength in the range of 450-750 nm was recorded during cooling at a temperature interval of 5 °C. An equilibration time of 5 mins was allowed at each temperature. The temperature was controlled by a Peltier device (Quantum Northwest, USA). Nuclear Magnetic Resonance (NMR). Two-dimensional 1H-1H nuclear Overhauser effect spectroscopy (2D 1H-1H NOESY) was recorded with a Bruker Avance NMR spectrometer operating 8


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at 500 MHz. Mixtures of 0.75%KC/0.75%gelatin in 99.9% D2O with addition of 0.1 M NaCl was prepared using the same dissolution method as described above. pH was adjusted to 7.0 using 1 M NaOD. The samples were heated at 85 °C for 10 mins and then sealed into NMR tube for analysis. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was added as an inner standard. A mixing time of 50 ms was applied to obtain the 2D correlation NMR spectra. The temperature was controlled by a Bruker BVT 3200 unit within ± 0.1 °C. Phase Compositional Analysis. Mixtures of 0.75%KC/0.75%gelatin at different temperatures and NaCl concentrations were subjected to phase compositional analysis after centrifugation at 10700 g (Pingfan, China). Different phases were carefully separated by using thin needle. Gelatin content in each phase was determined by the Micro-Kjeldahl method on its drying residues using an automatic apparatus (Hanon Instruments, China). A conversion factor of 5.55 was used for gelatin.33 KC content in each phase was calculated by subtracting the gelatin content from the total solid content. Average value of triplicate measurements was reported.

RESULTS AND DISCUSSION Phase Diagram in the Ionic Strength-Temperature Coordinate. The phase behavior of a 0.75%KC/0.75%gelatin aqueous mixture at pH 7.0 and various NaCl concentrations was investigated by turbidity, DSC and dynamic rheological measurement. The change in turbidity, heat flow, and storage and loss moduli as a function of temperature during cooling is shown in Figure 1. Heat flow profiles exhibit two exothermic peaks, with one being relatively broad and the other being sharp. The broad DSC peak has an onset transition temperature of ca. 22 °C, which is nearly independent of NaCl concentrations. The sharp DSC peak shows a strong dependence on salt concentration and moves to higher temperature with increasing NaCl concentration. By comparing 9



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to the DSC curves of the individual biopolymers, the broad and sharp DSC peaks can be readily attributed to the conformational transitions of gelatin and KC, respectively. Gelatin is well known to undergo a transition from random coil to triple helix during cooling, while that of KC is from random coil to double helix.20, 24 Salt type and concentration were reported to influence strongly the transition of KC,20, 23 which agrees with the present observation. When NaCl < 0.10 M, rheological results show a weak increase in G'' during cooling with G'' > G' in the whole temperature range examined. The increase in G'' coincides with the onset DSC transition temperature of gelatin, and therefore is caused by the formation of gelatin triple helices. G'' > G' suggests that the mixed systems are still in sol state even with gelatin triple helices.34 When

NaCl > 0.10 M, both G' and G'' display a clear increase during cooling and the increase coincides with the onset DSC transition temperature of KC. Moreover, G' was found to exceed over G'' at low temperatures. It indicates that the formation of KC double helices leads to the gelation of the mixed systems.29 The evolution of turbidity during cooling shows quite complex behaviors. When NaCl concentration is in the range of 0-0.15 M, the turbidity of KC/gelatin mixtures exhibits two turning points. At high temperatures, the turbidity is nearly constant, with a baseline value significantly higher than that of individual biopolymer solutions (data not shown). The appearance of turbidity at high temperatures is attributed to the associative phase separation of KC/gelatin induced by electrostatic interactions.8 Although being overall negatively charged, gelatin molecules at pH 7.0 possess locally positive patches that could interact with the negatively charged KC, resulting in the so-called complex coacervation of associative nature.8, 35 The electrostatically induced associative phase separation (EIAPS) is usually thought to be less temperature dependent,8, 36 and therefore shows a constant turbidity with lowering temperature. The first turning point of the turbidity displays 10


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a pronounced increase, starting at temperatures well above the conformational transitions of both KC and gelatin. Moreover, it shifts to higher temperatures when NaCl concentration is increased. Since individual KC or gelatin solution does not show any turbidity change during cooling in this range of salt concentration (data not shown), the first turbidity increase should arise from the interaction of KC with gelatin, rather than from the conformational transitions of the individual biopolymers. As will be discussed later, this turbidity change is assigned to a second type of associative phase separation induced by hydrogen bonding between KC and gelatin. With further lowering temperature, a second turning point appears in coincidence with the conformational ordering of KC. It implies that the pre-existing associative phase separations are modified by the formation of KC double

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Figure 1. Evolution of the turbidity at 500 nm (●), heat flow (—), storage (■) and loss (□) moduli as a function of temperature during cooling (0.5 °C/min) for 0.75%KC/0.75%gelatin aqueous mixtures at pH 7.0 and with varying NaCl concentrations: (a) 0 M; (b) 0.05 M; (c) 0.10 M; (d) 0.15 M; (e) 0.20 M; (f) 0.25 M; (g) 0.30 M; (h) 0.40 M; (i) 0.50 M; (j) 0.60 M. The upward DSC peaks represent exothermic processes.

In the NaCl concentration range of 0.15-0.25 M, the extent of the first turbidity increase for 12


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KC/gelatin mixtures is greatly reduced with increasing salt concentration. Moreover, it turns to shift to lower temperatures. The hydrogen bonding induced associative phase separation (HBIAPS) therefore has a maximum transition temperature at NaCl = 0.15 M. In contrast, the second turbidity increase becomes more and more pronounced. This turbidity increase is caused by the conformational ordering and gelation of KC, since individual KC solution in this salt concentration range exhibits a similar turbidity increase coinciding with its conformational transition. However, there is a possibility that segregative phase separation (SPS) between KC and gelatin could also contribute. It was previously reported that the conformational ordering in biopolymer mixtures such as KC/gelatin,7, 8 gelatin/maltodextrin,18 and gelatin/dextran,37 could induce SPS during cooling, which was attributed to an increased thermodynamic incompatibility between the biopolymers upon conformational transition.8, 13 The formation of KC double helices increases its molecular weight and unfavours the interaction with gelatin, leading to SPS. When NaCl concentration is > 0.25 M, there is a strong turbidity increase upon cooling, accompanied with the conformational ordering and gelation of KC. It moves to higher temperatures with increasing salt concentration. As discussed previously, this turbidity increase should be caused by the conformational ordering/gelation of KC, and possibly by the SPS between KC/gelatin as well. It should be pointed out that a slight decrease in turbidity was observed on further lowering temperature, and the decrease coincides with the conformational ordering of gelatin.

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1.5 1.0 0.5 0.0 0

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CNaCl (mol/L) Figure 2. Plot of the baseline value of turbidity at 60 °C obtained from Figure 1, as a function of NaCl concentration for 0.75%KC/0.75gelatin mixtures (○). The solid symbols (●) represent the sum of the turbidity from individual 0.75% KC and 0.75% gelatin solutions. As mentioned, the high baseline value of turbidity observed at high temperatures is attributed to the EIAPS between KC/gelatin. It is expected to be influenced by the addition of NaCl. Figure 2 plots the turbidity of 0.75%KC/0.75%gelatin mixtures measured at 60 °C as a function of NaCl concentration. The turbidity is reduced by increasing NaCl concentration up to 0.15 M. Afterward, it levels off and ends up with a value equal to the sum of the turbidity of individual biopolymer solutions. It manifests that the EIAPS is totally suppressed when NaCl > 0.15 M. This is due to a reduced electrostatic interaction screened by the addition of NaCl.8 Although no macroscopic phase separation occurs, soluble and compatible electrostatic complexes could still exist between KC and gelatin up to a certain NaCl concentration above 0.15 M.30 The complete suppression of EIAPS occurs at the same NaCl concentration (0.15 M) where the maximum transition temperature of HBIAPS is observed.

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0.7 0.6 (VII) SPS trapped by gelation

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Figure 3. Construction of phase diagram based on Figure 1 for 0.75%KC/0.75%gelatin aqueous mixtures at pH 7: the onset temperature of turbidity increase (●); the onset temperature of the conformational ordering of KC (+); the onset temperature of the conformational ordering of gelatin (× × ); the temperature below which no macroscopic phase separation could be observed after centrifuge (■). The phase diagram encompasses seven regions: I) compatible region; II) electrostatically induced associative phase separation (EIAPS) region; III) hydrogen bonding induced associative phase separation (HBIAPS) region; IV) coexistence of EIAPS and HBIAPS; V) segregative phase separation (SPS) region; VI) coexistence of HBIAPS and SPS; VII) SPS trapped by gelation. The horizontal solid line at NaCl = 0.15 M represents the boundary between compatible region and EIAPS. The horizontal solid line at NaCl = 0.25 M represents the upper boundary of HBIAPS. The arrows represents the typical salt concentrations (NaCl = 0.10, 0.20 and 0.40 M) at which phase compositional analysis was measured against temperature (corresponding to Figures 4, 5, and 6). The onset temperatures of turbidity increase and conformational orderings of KC and gelatin in 0.75%KC/0.75%gelatin mixtures are mapped in Figure 3. When NaCl > 0.25 M, the onset increase 15



of turbidity overlaps with the conformational ordering temperature of KC. When NaCl < 0.25 M, the onset increase of turbidity is well above the conformational ordering temperatures of both KC and gelatin. Moreover, the boundary between compatible region and EIAPS passes through the maximum of the onset temperatures of turbidity increase. Phase Behaviors Identified by Phase Compositional Analysis. In order to clarify the phase behaviors of KC/gelatin aqueous mixtures, macroscopic phase compositional analysis following centrifuge at typical NaCl concentrations and temperatures (indicated by the arrows in Figure 3) was conducted. Centrifugation at 10700 g was applied until equilibrium of macroscopic phase separation was attained, i.e., interfaces between different phases did not move with further prolonged centrifugation. (b)

100 80

Percentage (%)

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60 EIAPS + HBIAPS

40

EIAPS

20 0 0

10

20

30

40

50

60

70

T (oC)

Figure 4. Images (a) of the bulk phase separations after centrifugation for 0.75%KC/0.75%gelatin at NaCl = 0.10 M and various temperatures and the percent partitions (b) of KC and gelatin in the different phases as measured by phase compositional analysis: KC in upper phase (○); KC in lower phase (●); gelatin in upper phase (□); gelatin in lower phase (■). To guide the eye, red dashed lines were used to mark the interfaces in Figure (a).

Figure 4a shows the images of the bulk phase separations after centrifugation for 0.75%KC/0.75gelatin at NaCl = 0.10 M and various temperatures. Figure 4b is the percent partitions 16


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of KC and gelatin in the different phases. Clearly, two phases exist at temperatures higher than 40 °C. The upper phase is rather dilute and the lower phase is more like a swollen precipitate. The partitions of KC and gelatin in the upper phase are both ca. 90% and those in the lower phase are ca. 10%. The concentrated lower phase should be attributed to the complex coacervates formed between KC/gelatin as a result of EIAPS.8 When the temperature is below 40 °C the amount of the lower phase increases with decreasing temperature. The partitions of KC and gelatin in the lower phase increase, while those in the upper phase decrease. It means that more and more the biopolymers are concentrated in the lower phase when the temperature is lower than 40 °C. The transition is in concert with the observation of the turbidity increase in Figure 1c. Since EIAPS is known to be less temperature dependent,8, 36 the increased associative phase separation below 40 oC should arise from a second mechanism, which is of hydrogen bonding origin as will be discussed later. Therefore, the studied system at NaCl = 0.10 M and below 40 °C involves the coexistence of two types of associative phase separations induced by electrostatic interaction and hydrogen bonding. The coupling of hydrogen bonding and electrostatic interactions might lead to a metastable state of thermodynamic non-equilibrium. (b) 100 80

Percentage (%)

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60 HBIAPS + SPS

40

compatible

HBIAPS

20 0 0

10

20

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40

50

60

70

T (oC)

Figure 5. Images (a) of the bulk phase separations after centrifugation for 0.75%KC/0.75%gelatin at NaCl = 0.20 M and various temperatures and the percent partitions (b) of KC and gelatin in the 17



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different phases as measured by phase compositional analysis: KC in upper phase (○); KC in lower phase (●); KC in middle phase (∆); gelatin in upper phase (□); gelatin in lower phase (■); gelatin in middle phase (▲). To guide the eye, red dashed lines were used to mark the interfaces in Figure (a).

Figure 5a shows the images of the bulk phase separations after centrifugation for 0.75%KC/0.75%gelatin at NaCl = 0.20 M and various temperatures. When the temperature is higher than 35 °C, the mixtures after centrifugation are homogenous one-phase systems, and no phase separation is observed. KC and gelatin are thermodynamically compatible in this region. When the temperature is lower than 35 °C, phase separation results in two phases. The appearance of the phase separation is consistent with a slight turbidity increase around 35 °C as observed in Figure 1e. Interestingly, when the temperature is further decreased below 20 °C, the mixed systems display a middle phase in addition to the upper and lower phases. Figure 5b shows the corresponding percent partitions of KC and gelatin in each phase. At temperatures higher than 35 °C, the mixtures are compatible one-phase systems, and therefore KC and gelatin are both 100% present in the upper phase (no lower phase). In the temperature range of 20-35 °C, the partitions of KC and gelatin in the lower phase increase with decreasing temperature, while those in the upper phase decrease. This behavior indicates an associative nature of the phase separation, and is similar to the transition observed below 40 °C at NaCl = 0.10 M (Figure 4). It is attributed to the HBIAPS between KC and gelatin. Since no significant electrostatic interaction or conformational ordering exists under those conditions, HBIAPS here represents a thermodynamic equilibrium state. At temperatures below 20 °C, the partitions of KC and gelatin in the lower phase do not change significantly. However, due to the formation of the middle phase, the partitions of KC and gelatin in the upper phase are dramatically reduced around 20 °C. Moreover, KC and gelatin are

18


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unevenly distributed in the upper and middle phases. Specifically, the partition of KC in the middle phase (ca. 69%) is much higher than that in the upper phase (ca. 12%). On the other hand, the partition of gelatin in the middle phase (ca. 15%) is much lower than that in the upper phase (ca. 75%). The enrichment of KC and gelatin in the separate phases indicates a segregative nature of the phase separation.5, 6 The transition around 20 °C roughly coincides with the conformational ordering of KC. It is clear that a segregative phase separation (SPS) is induced on top of the HBIAPS when KC transforms into double helical conformation. SPS should occur to the dilute phase of HBIAPS, so that the concentrated (lower) phase is almost unchanged. It is conceivable that the gelation of KC in the concentrated phase (as shown in Figure 1e) kinetically traps the HBIAPS and makes the coexistence of SPS+HBIAPS possible.5, 6 The coexisting state therefore should be a thermodynamic non-equilibrium state. (b)

100 80

Percentage (%)

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60

SPS trapped by 40 gelation

SPS

compatible

20 0 0

10

20

30

40

50

60

70

T (℃ ℃)

Figure 6. Images (a) of the bulk phase separations after centrifugation for 0.75%KC/0.75%gelatin at NaCl = 0.40 M and various temperatures and the percent partitions (b) of KC and gelatin in the different phases as measured by phase compositional analysis: KC in upper phase (○); KC in lower phase (●); gelatin in upper phase (□); gelatin in lower phase (■). To guide the eye, red dashed lines were used to mark the interfaces in Figure (a).

Figure 6a shows the images of the bulk phase separations after centrifugation for 19



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0.75%KC/0.75%gelatin at NaCl = 0.40 M and various temperatures. The mixtures above 30 °C are homogenous one-phase systems, whereas those below 30 °C display two phases. The partitions of KC and gelatin shown in Figure 6b also exhibit a clear transition around 30 °C. At temperatures higher than 30 °C, KC and gelatin are 100% present in the upper phase, since there is no lower phase. At intermediate temperatures below 30 °C, the partition of KC in the lower phase (ca. 84%) is much higher than that in the upper phase (ca. 16%). On the contrary, the partition of gelatin in the upper phase (ca. 74%) is much higher than that in the lower phase (ca. 26%). This suggests that KC and gelatin are separately concentrated in the lower and upper phases, indicating a segregative nature of the phase separation. Since the transition is very close to the conformational ordering temperature (33 °C) of KC at NaCl = 0.40 M, the SPS is induced by the formation of KC double helices. Intriguingly, with further decreasing temperature below 15 °C, the partitions of KC and gelatin again change markedly, although the systems remain two phases. The partitions of KC and gelatin in the upper phases almost decrease to 0%, while those in the lower phase are nearly 100%. The watery upper phase is found to be a result of syneresis of the gelled mixtures when subjected to centrifugation. Gelation in this region is strong enough to trap SPS to prevent macroscopic phase separation, resulting in a metastable state of thermodynamic non-equilibrium. The transition marks a state of SPS trapped by gelation, and was found to move to higher temperature with increasing NaCl concentration (square symbols in Figure 3). The reason is due to an increased gelling ability when salt concentration is increased.20, 23 Phase compositional analysis shown above identified complex phase behaviors for 0.75%KC/0.75%gelatin in relation to the diagram drawn in Figure 3: I) compatible region; II) EIAPS region; III) HBIAPS region; IV) coexisting region of EIAPS+HBIAPS; V) SPS region; VI) coexisting region of HBIAPS+SPS; and VII) region of SPS trapped by gelation. The 20


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thermodynamically compatible mixtures exist at higher temperatures than the conformational ordering temperatures of KC and at higher NaCl concentrations, i.e., NaCl > 0.15 M. The EIAPS occurs at lower NaCl concentrations (< 0.15 M), where electrostatic interaction prevails. The HBIAPS could occur at relatively low NaCl concentration and even on top of the EIAPS. The starting temperature of HBIAPS is well above the conformational ordering temperatures of KC and gelatin. The SPS is induced by the conformational ordering of KC due to an increased incompatibility between KC and gelatin upon KC helix formation. At lower temperatures and higher NaCl concentrations, the gelation of the mixtures is strong enough to trap SPS and prevents macroscopic phase separation. It should be pointed out that the three coexisting regions, i.e. IV, VI and VII, should represent metastable states where thermodynamic equilibriums are not reached due to the entrapment by gelation or complexation. Different pathways of entering the coexisting regions, e.g. approaching from a fixed NaCl concentration or a fixed temperature may generate different final states. This interesting property will be demonstrated in future studies. Mechanism of HBIAPS. As described previously, the present work observed for the first time a turbidity increase upon cooling for KC/gelatin mixtures at temperatures much higher than their conformational ordering temperatures (Figures 1a-1f). The phenomenon is only present when NaCl concentration is in the range of 0-0.25 M, and shows a maximum transition temperature at NaCl = 0.15 M (Figure 3). Phase compositional analysis demonstrates an associative phase separation accompanying the turbidity increase (Figures 4 and 5). Here we will show that this is a new type of associative phase separation induced by hydrogen bonding, and is different from the conventional electrostatically induced associative phase separation (also known as complex coacervation).

21



6 5

τ (cm-1), Dh (µm)

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4 3 2 1 0 0

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60

70

T (oC) Figure 7. Evolution of the turbidity at 500 nm (—) and the z-average hydrodynamic diameter Dh (●) as a function of temperature for the soluble fraction of 0.75%KC/0.75%gelatin mixture in the presence of 0.10 M NaCl, as obtained by centrifugation at 70 °C. Due to the measuring limits of ZetaSizer Nano-ZS, the data for Dh > 6µm was not shown.

To separate the contribution of complex coacervate, the mixture of 0.75%KC/0.75%gealatin at NaCl = 0.10 M was subjected to centrifuge at 70 °C to remove coacervate phase. The remaining soluble phase was examined for the evolution of turbidity and particle size as a function of temperature. It can be seen from Figure 7 that the soluble fraction exhibits a similar transition at 42 °C, as is observed for that without centrifugation (Figure 1c). Moreover, the hydrodynamic diameter of the soluble fraction grows significantly at 42 °C. It suggests that this transition is not caused by the re-arrangement of the coacervate phase, but instead it is attributed to a second event associated with the soluble fraction. This is consistent with the fact that the transition could also be observed in the NaCl concentration range of 0.15-0.25 M for compatible mixtures where no EIAPS (i.e. complex coacervation) is present (Figure 3).

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5

control 0.2M urea 0.5M urea 1.0M urea 2.0M urea 1.0M glycerol 2.0M glycerol

4

τ (cm-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3 2 1 0 0

10

20

30

T

40

50

60

70

(oC)

Figure 8. Effect of urea and glycerol on the turbidity change during cooling (0.5 °C/min) for 0.75%KC/0.75%gelatin in the presence of 0.10 M NaCl.

Figure 8 shows the effect of urea and glycerol on the transition as reflected by the turbidity change during cooling for 0.75%KC/0.75%gelatin in the presence of 0.10 M NaCl. It is well known that urea is a hydrogen bond breaker and glycerol is a hydrogen bond promoter.38, 39 Compared to the control, the addition of urea greatly reduces the extent of the turbidity increase. Moreover, the transition temperature shifts to lower values with increasing urea concentration. The transition disappears completely at 2.0 M urea. On the contrary, the addition of glycerol slightly increases the transition temperature. These results indicate that the transition should be of hydrogen bonding origin. With lowering temperature, hydrogen bonds are formed between KC and gelatin, which increases their attractive interaction and induces a second type of associative phase separation (namely, HBIAPS). Methylene blue assay was used to probe the interaction of KC/gelatin as a function of temperature during cooling. The UV/Vis absorption spectra of KC and MB mixture at NaCl = 0.10 M are shown in Figure 9a. The absorption at 554 nm arises from the parallel and close binding of MB molecules 23



along KC chain. The absorption at 663 nm arises from free and unbound MB molecules. It is supposed that the interactions between KC/gelatin would alter the number of MB molecules bound to KC. Figure 9b plots the absorbance difference ∆A at 554 and 663 nm between the systems with and without addition of gelatin, as a function of temperature. ∆A at 554 and 663 nm both show a transition around 40 °C, which is very close to the temperature of turbidity increase as observed in Figure 1c. 70℃ 60℃ 50℃ 40℃ 30℃ 20℃ 10℃

1.2

(b)

65℃ 55℃ 45℃ 35℃ 25℃ 15℃ 5℃

0.36 554nm 663nm

0.24

△A

1.6

(a)

A

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0.8

0.4

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0.00

0

-0.12

450

500

550

600

650

700

750

0

10

20

30

40

50

60

70

T (oC)

λ (nm)

Figure 9. Absorption spectra of KC/MB mixture at various temperatures (a) and absorbance difference ∆A at 554 and 663 nm between KC/gelatin/MB and KC/MB (b), plotted against temperature. ∆A=AKC/gelatin/MB- AKC/MB. NaCl = 0.10 M.

When the temperature is above 40 °C, ∆A at 554 nm is constant and nearly zero, indicating the presence of gelatin has limited effect on the number of MB molecules bound to KC. As discussed previously, gelatin and KC interact electrostatically in this region (Figure 3). It implies that the electrostatic association of gelatin on to KC hardly occupies the binding sites of MB. A possible explanation is that the long-range electrostatic interaction only results in a loose binding of gelatin with KC,40 and is not effective in occupying the consecutive binding sites (i.e. sulfate groups) along KC chain that are crucial for the formation of MB metachromatic complexes. With further decreasing temperature below 40 °C, ∆A at 554 nm becomes more and more negative. 24


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It suggests a decreasing number of MB molecules in bound state during the transition. The decrease in bound MB should be caused by the formation of hydrogen bonds between KC and gelatin below 40 °C. On the other hand, ∆A at 663 nm shows an increase below 40 °C. It indicates an increasing number of free MB molecules during the transition. This is in harmony with the data at 554 nm. As a short-range interaction,40 hydrogen bonding can bring KC and gelatin into close contact, and result in possible alignments of KC and gelatin segments via hydrogen bonding network. This configuration seriously reduces the consecutive binding sites along KC chain that are available for MB molecules. The possible interacting sites between KC and gelatin associated with the transition were studied by NOESY measurements. The NOESY spectrum provides distance information between pair of hydrogen atoms separated by less than 0.5 nm.41, 42 Figure 10 is the 2D 1H-1H NOESY spectra of 0.75%KC/0.75%gelatin in the presence of 0.10 M NaCl recorded at 60 °C and 30 °C, respectively. A conspicuous intermolecular cross-peak is observed if the temperature is below the transition temperature of 42 °C (Figure 10b), while the cross-peak is absent above the transition temperature (Figure 10a). This indicates that gelatin is spatially close to KC within a distance of 0.5 nm below the transition temperature. The proximity is comparable to the packing distance of MB molecules (0.35-0.77 nm) in KC/MB metachromatic complexes,32 and therefore can disrupt the binding of MB to KC as revealed in Figure 9b.

25



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Figure 10. 2D 1H-1H NOESY spectra of 0.75%KC/0.75%gelatin mixture in the presence of 0.10 M NaCl at 60 °C (a) and 30 °C (b), respectively. Based on literature data,43-45 the cross-peak is assigned to the C6/C5 protons of the α-(1-3)-D-galactose-4-sulfate residue of KC (δ = 3.75 ppm) and the β, γ, δ-CH2 protons of the proline residue of gelatin (δ = 2.10 ppm). This indicates that the hydrogen bonds occur between the two residues specifically. Since galactose-sulfate and proline are the major repeating units of KC and gelatin,44, 46 it means that they could form extensive hydrogen bonds (or even hydrogen bonding network) by side-by-side alignment of the biopolymer chains, which eventually leads to HBIAPS. It should be pointed out that in gelatin triple helix the proline residue is also believed to play an important stabilizing effect by participating in inter-chain hydrogen bonding formation.47

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Moreover, the transition temperature of HBIAPS lies in the range of ca. 30-45 °C (Figure 3), which is to some extent higher than the onset conformational ordering temperatures of gelatin and KC. Gelatin triplex is known to melt in the range of 30-40 °C, whereas its formation needs a certain extent of supercooling.24 This has been attributed to the requirement of nucleation process before the growth of triple helix. It is reasonable that within this particular temperature window, the local structural arrangement of gelatin or the local change in solvent environment upon cooling,24, 48 allows the formation of hydrogen bonds with KC and consequently the HBIAPS. HBIAPS between KC/gelatin is reminiscent of the simple coacervation of gelatin itself in a mixed solvent of water-alcohol, where hydrogen bonding is also believed to be a dominating molecular force.49 Furthermore, NaCl seems to have dual effects on HBIAPS. The addition of NaCl up to 0.15 M promotes the HBIAPS, while a further addition of NaCl turns to suppress it (Figure 3). A maximum transition temperature of HBIAPS is observed at NaCl = 0.15 M. This salt concentration also corresponds to the boundary between EIAPS and compatible region (Figure 3). The promotive effect of NaCl can be explained as: 1) the presence of small amount of NaCl facilitates the electrostatic complexation of KC/gelatin by screening long-range electrostatic repulsion,30 which in turn helps the subsequent formation of hydrogen bonds during cooling by bringing the biopolymers into close contact; 2) the addition of NaCl changes the solvent quality and hence the hydration of the biopolymers, and promote the hydrogen bonding between them. The suppressive effect of NaCl can be explained by that a further addition of NaCl tends to dissociate the electrostatic complexes of KC/gelatin, which impairs the subsequent formation of hydrogen bonds. The electrostatic complexation at low ionic strength is therefore an advantage to the occurrence of HBIAPS.

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CONCLUSIONS In conclusion, we have studied the complex phase behavior of aqueous mixture of oppositely charged gelling biopolymers, containing KC and type B gelatin. A detailed and complete phase diagram was constructed in the ionic strength-temperature coordinate, by monitoring conformational transitions, rheological and turbidity changes as well as bulk phase separations accelerated via centrifugation. The interplay of biopolymer conformational orderings with different molecular forces such as electrostatic interaction and hydrogen bonding generated rich and diverse phase behaviors for the mixture, which included: I) compatible mixture; II) EIAPS; III) HBIAPS; IV) coexistence of EIAPS and HBIAPS; V) SPS; VI) coexistence of HBIAPS and SPS; VII) SPS trapped by gelation. SPS was found to be related to the conformational ordering of KC during cooling. HBIAPS occurred as a result of extensive hydrogen bonding between KC and gelatin prior to their conformational orderings. Methylene blue spectrophotometry and NMR revealed specifically that the galactose-sulfate residues of KC and the proline residues of gelatin are spatially close and are responsible for the formation of hydrogen bonds between the biopolymer chains. NaCl has dual effects (promotive vs. suppressive) on HBIAPS and an optimum concentration was observed at NaCl = 0.15 M. The pre-formation of electrostatic complexes between KC and gelatin at low NaCl concentrations facilitated the subsequent formation of hydrogen bonds upon cooling, therefore favoring HBIAPS. The in-depth understanding of the complex phase behaviors of aqueous mixture of oppositely charged gelling biopolymers, gained in the study, is of technological importance for the applications of biopolymer mixtures in different industries, e.g., creating new microstructures or mechanical properties.

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ACKNOWLEDGMENTS The research was supported by National Natural Science Foundation of China (31322043, 31171751), Projects from Hubei Provincial Department of Science and Technology (2014BHE004, 2012FFA004) and Department of Education (T201307), Program for New Century Excellent Talents in University (NCET-12-0710), and Key Project of Chinese Ministry of Education (212117).

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TOC graphic: 0.7 0.6 (VII) SPS trapped by gelation

0.5

CNaCl (mol/L)

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0.4

(V) SPS

(I) compatible

0.3 0.2

(VI) HBIAPS+SPS

(III) HBIAPS

0.1

(IV) EIAPS+HBIAPS

(II) EIAPS

0.0 0

10

20

30

40

50

60

T (oC)

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Mapping the Complex Phase Behaviors of Aqueous Mixtures of Îº

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