International Journal of Biological Macromolecules 79 (2015) 894–902

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Characterization of fish gelatin–gum arabic complex coacervates as influenced by phase separation temperature Mohammad Anvari a,b , Cheol-Ho Pan c,d , Won-Byong Yoon e , Donghwa Chung f,g,∗ a

Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung 210-702, Republic of Korea School of Food Science, University of Idaho, Moscow, ID 83843, USA Laboratory of Biomodulation, KIST Gangneung Institute of Natural Products, Gangneung 210-340, Republic of Korea d Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon 305-350, Republic of Korea e Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea f Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea g Institute of Food Industrialization, Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 29 March 2015 Received in revised form 30 May 2015 Accepted 2 June 2015 Available online 6 June 2015 Keywords: Fish gelatin–gum arabic complex coacervation Phase separation temperature Oscillatory shear

a b s t r a c t The rheological and structural characteristics of fish gelatin (FG)–gum arabic (GA) complex coacervate phase, separated from an aqueous mixture of 1% FG and 1% GA at pH 3.5, were investigated as influenced by phase separation temperature. Decreasing the phase separation temperature from 40 to 10 ◦ C lead to: (1) the formation of a coacervate phase with a larger volume fraction and higher biopolymer concentrations, which is more viscous, more structural resistant at low shear rates, more shear-thinning at high shear rates, and more condensed in microstructure, (2) a solid-like elastic behavior of the phase separated at 10 ◦ C at a high oscillatory frequency, (3) the increase in gelling and melting temperatures of the coacervate phase (3.7−3.9 ◦ C and 6.2−6.9 ◦ C, respectively), (4) the formation of a more rigid and thermo-stable coacervate gel. The coacervate phase is regarded as a homogeneously networked biopolymer matrix dispersed with water vacuoles and its gel as a weak physical gel reinforced by FG–GA attractive electrostatic interactions. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Protein–polysaccharide interactions in an aqueous environment have been extensively studied in recent years because of their high potential applications in the development of bioactive delivery devices, fat replacers, meat analogs, gels, emulsions, edible films, and coatings in food and pharmaceutical industries [1–3]. The interactions are either attractive or repulsive, depending on biopolymer characteristics (e.g., molecular weight, charge density, conformation, and flexibility), solvent properties (e.g., pH and ionic strength), and mixing conditions (e.g., protein to polysaccharide ratio, total biopolymer concentration, temperature, stirring, and pressure) [4,5]. Attractive interactions, primarily attributed to electrostatic attractions between oppositely charged biopolymers, induce the formation of biopolymer complexes, which could be either soluble in a single phase or insoluble to build a

∗ Corresponding author at: Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea. Tel.: +82 33 339 5793; fax: +82 33 339 5716. E-mail address: [email protected] (D. Chung). http://dx.doi.org/10.1016/j.ijbiomac.2015.06.004 0141-8130/© 2015 Elsevier B.V. All rights reserved.

two-phase system where one phase is enriched in insoluble complexes and the other depleted [3,6]. The sequential process, composed of (1) the formation of charge neutralized insoluble complexes and (2) the subsequent spontaneous macroscopic phase separation to complex-rich and solvent-rich phases, is known as associative separation, which can be also called complex coacervation or precipitation for the separation of liquid-state insoluble complexes (complex coacervates) or solid-state insoluble complexes (precipitates), respectively [3,6]. The formation of insoluble protein–polysaccharide complexes can be significantly influenced by temperature through the temperature-dependent alterations not only in the conformation of biopolymers but also in the noncoulombic interactions between the biopolymers, such as hydrogen bonding and hydrophobic interactions. Harding et al. [7] demonstrated that the conformational change of bovine serum albumin by heat-induced denaturation enhanced the formation of insoluble complexes with alginate. Fang et al. [8] showed that the conformational change of pigskin gelatin by heat-induced transition from helix to random coil could also enhance the complexation with ␬-carrageenan. Hydrogen bonding, in principle, is favored at low temperatures, whereas hydrophobic interactions become stronger with increasing temperature,

M. Anvari et al. / International Journal of Biological Macromolecules 79 (2015) 894–902

because of the exposure of more interaction sites by heat-induced conformational changes of the biopolymers [1,9,10]. Liu et al. [11] also reported that a decrease of temperature from 23 to 6 ◦ C significantly improved the complexation between pea protein isolate and gum arabic by strengthening hydrogen bonding as a secondary force, whereas hydrophobic interactions appeared to be more prevalent in a higher temperature range of 23−60 ◦ C and to be associated with the stability, rather than the formation of complexes. The spontaneous macroscopic phase separation of insoluble complexes may be also strongly dependent on temperature. The phase separation phenomenon, mainly driven by entropy, takes place by a nucleation-and-growth-like mechanism, as a result of the rearrangement, coalescence, Ostwald ripening, or aggregation of insoluble complexes, mostly via the reformation of intermolecular interactions and the expulsion of water molecules [1,12,13]. It was suggested by Nigen et al. [14] that the initial complexation between two oppositely charged globular proteins, ␣-lactalbumin and lysozyme, might be caused by electrostatic attractions, however, the growth of complexes could be largely driven by hydrogen bonding at low temperatures, resulting in the formation of reversible aggregates, but by hydrophobic interactions at higher temperatures to form coacervate-like structures. Kayitmazer et al. [15] showed that the shear-dependent viscosity and dynamic moduli of bovine serum albumin-chitosan coacervate phase was significantly dependant on temperature, although the temperature was not the phase separation temperature but the temperature of measurements. It is therefore expected that the rheology and structure of complex-rich phase obtained by protein–polysaccharide complex coacervation and the sol–gel transformation of such coacervate phase, which are directly related to the success of some valuable applications such as microencapsulation and emulsion stabilization, could be considerably influenced by the temperature of phase separation. However, such aspects of complex coacervation have not been systematically studied. Fish gelatin (FG) is regarded as a promising alternative to mammalian gelatins, because it can be produced from fish skin and bones, comprising nearly 30% of fish processing byproducts, and does not have safety- or religion-related consumer concerns [3]. It was found by our group that the FG from cold water fish skin (58 kDa) underwent complex coacervation with gum arabic (GA, 382 kDa), one of the most widely used anionic polysaccharides, in aqueous solutions at 40 ◦ C [3]. The formation of insoluble or soluble FG–GA complexes was strongly influenced by pH and FG to GA weight ratio, as often reported for other protein–anionic polysaccharide pairs, and the total concentration and composition of biopolymers in the coacervate phase was dependent on the total concentration of biopolymers in the initial mixture. In the present study, we aimed to investigate the rheological and structural characteristics of FG–GA complex coacervate phase as influenced by phase separation temperature, using dynamic oscillatory and steady shear rheological measurements, Fourier transform infrared spectroscopy (FTIR), confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM).

2. Materials and methods 2.1. Materials Fish gelatin (FG, from cold water fish skin) and gum arabic (GA, from Acacia tree) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and Carl Roth GmbH (Karlsruhe, Germany), respectively. The average molecular weights of FG and GA, determined by viscometry using an Ostwald viscometer and Mark–Houwink equation in our preliminary experiments, were 58 and 382 kDa,

895

respectively [3]. Sodium azide (NaN3 ) was obtained from Daehung Chemicals & Metals (Siheung, Korea). Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade purity. 2.2. Preparation of FG–GA coacervate phase Accurately weighed amounts of FG and GA were separately dissolved in 100 mL of distilled water at 40 ◦ C and mixed together at a weight ratio of FG to GA (FG:GA) of 1:1 and a total biopolymer concentration of 2% (w/v), followed by adjusting the pH to 3.5. Sodium azide was added as a preservative at a concentration of 0.02% (w/v). The FG–GA mixture was incubated at 40 ◦ C in a shaking water bath at 100 rpm for 24 h for sufficient electrostatic interactions between FG and GA molecules, and then placed statically at 10, 30, or 40 ◦ C for another 24 h for the macroscopic phase separation of FG–GA insoluble complexes to reach equilibrium state. 2.3. Measurements of phase volume and composition The volume fraction of coacervate phase was determined as follows: Volume fraction (%) =

Vc × 100 Vt

(1)

where Vc is the volume of coacervate phase and Vt is the total volume of FG–GA mixture. The amounts of total biopolymers and FG in solvent-rich phase were measured by oven drying at 105 ◦ C for 24 h and by the Lowry method, respectively [3,16]. The values measured above were used to calculate the amount and concentration of biopolymers in the coacervate phase and the FG:GA value of coacervate phase. All measurements were performed at least in triplicate. 2.4. Fourier transformed infrared (FTIR) spectroscopy FTIR spectroscopy was used to examine the changes in the secondary structure of FG and molecular interactions between biopolymers, such as electrostatic interactions, hydrogen bonding, or hydrophobic interactions, in the coacervate phase. The coacervate phases obtained were dried in a vacuum oven at their phase separation temperatures. Aqueous solutions of FG and GA, separately prepared at 1% (w/v) and pH 3.5, were also dried as controls. The dried samples of 2 mg in approximately 100 mg potassium bromide were placed in discs, and FTIR spectra were obtained in the wavenumber range of 4000–500 cm−1 using an infrared spectrophotometer (Tensor 27, Bruker Instruments, Billerica, MA, USA) at a scanning rate of 10 kHz with 64 scans and a resolution of 4 cm−1 . The background was obtained against pure potassium bromide pellet. All data treatments were carried out using OPUS 7.0 software (Bruker Instruments, Billerica, MA, USA). 2.5. Confocal scanning laser microscopy (CSLM) CSLM was used to examine the microstructure of FG–GA coacervate phase. FG and GA were covalently stained with fluorescent markers, FITC and RITC, respectively. The excitation/emission wavelengths of FITC and RITC are 485/530 nm and 530/590 nm, respectively. The staining was performed as described in Schmitt et al. [9]. Briefly, aqueous solutions of FG and GA were separately prepared at 2% (w/v), and their pH values were adjusted to 8.5 and 10.5, respectively, to facilitate the staining reaction. Thereafter, 25 ␮L of 2% (w/w) FITC or RITC solution in DMSO was added to 100 mL of the FG or GA solution, respectively, and the staining reaction was allowed to proceed for 1.5 h with gentle stirring at

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Table 1 Volume fraction and composition of FG–GA coacervate phasesa separated at three different temperatures. Parameter

Phase separation temperature 40 ◦ C

Volume fraction (%, v/v) FG content (mg) GA content (mg) Total biopolymer content (mg) FG concentration (%, w/v) GA concentration (%, w/v) Total biopolymer concentration (%, w/v) FG to GA weight ratio (FG:GA)

7.0 233.3 170.5 403.8 8.3 6.1 14.4

30 ◦ C ± ± ± ± ± ± ±

b

0.1 1.8b 1.3c 1.9c 0.0a 0.0c 0.1b

1.4 ± 0.0a

7.1 235.2 174.9 410.0 8.3 6.2 14.4

10 ◦ C ± ± ± ± ± ± ±

b

0.3 1.3b 0.8b 2.5b 0.0a 0.0b 0.1b

1.3 ± 0.0a

7.8 239.0 231.0 470.0 7.7 7.4 15.1

± ± ± ± ± ± ±

0.1a 0.8a 2.0a 1.7a 0.0b 0.0a 0.1a

1.0 ± 0.1b

a

All the phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C. Values are expressed as mean ± standard deviation of at least three different samples. Values with different superscript letters in the same row are significantly different at p < 0.05.

40 ◦ C. The two solutions were then mixed together as described in Section 2.2 to obtain FG–GA coacervate phases separated at three different temperatures (10, 30, and 40 ◦ C). The microstructure of stained FG–GA coacervate phases were imaged using FV-300 confocal scanning unit (Olympus, Tokyo, Japan) equipped with an Olympus IX71 inverted microscope and an argon ion laser. The laser was adjusted in the green/red fluorescence mode, yielding two excitation wavelengths of 485 and 530 nm, to obtain green and red fluorescence images from two separate channels. All images were taken at a magnification of 40× (oil immersion, numeric aperture 1.30) and processed using Fluoview Software (Olympus, Tokyo, Japan). 2.6. Scanning electron microscopy (SEM) SEM was also used to examine the microstructure of the FG–GA coacervate phases separated at three different temperatures (10, 30, and 40 ◦ C). Each sample was fixed in 2% (w/w) glutaraldehyde solution, dehydrated using different aqueous ethanol solutions, and placed in acetone as described in Espinosa-Andrews et al. [17]. Each sample was dried in a vacuum oven for 5 min, fragmented, and mounted on stubs with the fractured face upwards, and gold-coated with a fine coat ion sputter (JFC 1100, JEOL Ltd., Akishima, Japan). A low vacuum scanning electron microscope (Inspect F50, FEI Co., Hillsboro, OR, USA), was used at 15 kV to obtain the SEM images of the cross-section of each sample at magnifications of 100× and 1000×, respectively. 2.7. Rheological analysis All rheological measurements were carried out using a TAAR 2000 rheometer (TA Instruments Inc., New Castle, DE, USA), equipped with a cone–plate geometry (diameter 40 mm, angle 2◦ , and gap 53 ␮m) and a moisture trap. Firstly, the steady shear behavior of the FG–GA coacervate phases separated at three different temperatures (10, 30, and 40 ◦ C) was examined by measuring the apparent viscosity (a ) of each phase as a function of shear rate (). ˙ Each sample was sheared from 0.01 to 100 1/s at its phase separation temperature, during which 61 data points were recorded at 6 s intervals. As controls, three aqueous mixtures of FG and GA were prepared with the same biopolymer compositions as the coacervate phases, determined in Section 2.3 (Table 1), at pH 8.0, where there are no significant attractive interactions between FG and GA.

Secondly, the dynamic oscillatory behavior of the three FG–GA coacervate phases was examined by frequency sweep tests, where the elastic or storage modulus (G ) and viscous or loss modulus (G ) of each phase were determined as a function of angular frequency (ω) in the range of 0.1–100 rad/s. The linear viscoelastic region, where G and G are independent of strain (), was determined by strain sweep tests in the  range of 0.01–100% and at ω = 2 rad/s. For the coacervate phases separated at 10, 30, and 40 ◦ C, the linear viscoelastic region was found to be up to 2.0%, 1.3%, and 1.2%, respectively (data not shown), and therefore, a  value of 1% was used for the frequency sweep and temperature sweep tests. Thirdly, the sol–gel transformation of the three FG–GA coacervate phases was investigated by temperature sweep tests, where G and G were monitored at ω = 2 rad/s and  = 1% with decreasing temperature from each phase separation temperature to 3 ◦ C and then back to the phase separation temperature at a cooling or heating rate of 0.016 ◦ C/min, and the gelling and melting points (Tg and Tm , respectively) of each coacervate phase were determined. Complex modulus (G*) was obtained at 3 ◦ C and used to compare the overall rigidity of the gels transformed from the coacervate phases. G∗ =



(G )2 + (G )2

(2)

Finally, the dynamic oscillatory behavior of the gels obtained by cooling the coacervate phases to 3 ◦ C was also examined by frequency sweep tests at  = 1% in the range of 0.1–100 rad/s. All the rheological measurements were performed at least in triplicate.

3. Results and discussion 3.1. Volume fraction and composition of FG–GA coacervate phase The volume fraction of FG–GA coacervate phase was found to increase from 7.0% to 7.8% with decreasing phase separation temperature from 40 to 10 ◦ C (Table 1). The values were close to the value (about 8.0%) reported for the coacervate phase separated from the mixture of gelatin (unknown source) and GA, which was prepared under similar conditions as in the present study: a weight ratio of gelatin to GA of 1:1, a total biopolymer concentration of 2%, pH about 3.5, and a mixing and phase separation temperature of 40 ◦ C [18]. The composition analysis shows that the amount and concentration of total biopolymer and GA in the coacervate phase, as well as the amount of FG in the phase significantly increased with decreasing phase separation temperature, while the concentration of FG was reduced from 8.3% to 7.7% (w/v), resulting in the considerable decrease in the weight ratio of FG to GA (FG:GA) from 1.4 to 1.0 (Table 1). The results indicate that decreasing phase separation temperature from 40 to 10 ◦ C not only increase the volume fraction of coacervate phase, but also enhance the partition of the two biopolymers, especially GA molecules, into the coacervate phase. The volume increase of coacervate phase may be partly attributed to the partition of more GA molecules (382 kDa), which are much larger than FG molecules (58 kDa), into the phase at lower phase separation temperature [13]. The phase separation of protein–polysaccharide mixture is known to be an entropy-driven process occurring through a nucleation-and-growth like mechanism, where the biopolymer complexes formed during the mixing undergo rearrangement, coalescence, Ostwald ripening, or aggregation, mostly via the reformation of intermolecular interactions and the expulsion of water molecules [1,12,13]. Different phase volumes and compositions of FG–GA coacervate phases separated at different temperatures suggest that temperature could be an important factor influencing the phase separation mechanism of FG–GA complexes.

M. Anvari et al. / International Journal of Biological Macromolecules 79 (2015) 894–902

3.2. Fourier transformed infrared (FTIR) spectroscopy

897

10 o

FG-GA at 40 C

amide I amide II

amide A

FG 40 oC 1635 1521

3429

FG 30 oC 1633

3427

1521

o

Transmittance (%)

FG 10 C

1541 1656

o

3423

GA 40 C 1612 1423

3440

o

FG-GA at 30 C o

FG-GA at 10 C Control at 40 oC

1

Control at 30 oC

ηa (Pa.s)

Fig. 1 shows the FT-IR spectra of FG, GA, and the FG–GA coacervate phases separated at three different temperatures. The changes in the secondary structure of FG and the molecular interactions between biopolymers, such as electrostatic interactions and hydrogen bonding, in the coacervate phases were investigated by examining three band regions: amide A (3600–2300 cm–1 ) for N H stretching, amide I (1600–1700 cm–1 ) for primarily C O stretching, and amide II (1335–1560 cm–1 ) for N H bending and C N stretching [18–21]. At 40 ◦ C, FG molecules showed a typical amide I peak at 1635 cm−1 , which is regarded as a characteristic for the random coil structure of gelatin [19,22,23], and GA molecules showed two peaks at 1612 and 1423 cm−1 due to the asymmetric and symmetric stretching of carboxyl acid salt COO− , respectively (Fig. 1) [17]. The FG–GA coacervate phase obtained at 40 ◦ C did not show the two GA peaks, implying the involvement of COO− of GA molecules in electrostatic interactions, and its amide I peak at 1632 cm−1 indicates that the FG molecules complexed with GA molecules also had a random coil structure at 40 ◦ C. At 30 ◦ C, FG showed a similar spectrum to that at 40 ◦ C, while the FG–GA coacervate phase exhibited an amide A peak at a lower wavenumber (3420 cm−1 ) and an amide II peak at a higher wavenumber (1539 cm−1 ) (Fig. 1). Amide A and amide II bands are attributed to the stretching vibration of N H group bonded to O H group and the C N stretching combined with N H deformation of peptide group, respective, and therefore, both the wavenumber

Control at 10 oC Carreau fitting curve

0.1

0.01 0.01

0.1

1

10

100

.

γ (1/s) Fig. 2. Apparent viscosity (a ) versus shear rate () ˙ for FG–GA coacervate phases separated at 10, 30, or 40 ◦ C and their corresponding controls. The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C. The controls are the FG–GA mixtures prepared with the same biopolymer compositions as the coacervate phases, but at pH 8.0.

downshift of amide A peak and the upshift of amide II peak suggest the increase of hydrogen bonding in the coacervate phase [20,24]. The FG molecules in the coacervate phase may also have a random coil structure at 30 ◦ C, considering the amide I peak at 1639 cm−1 . When the temperature was further reduced to 10 ◦ C, FG showed amide I and amide II peaks at higher wavenumbers (1656 and 1541 cm−1 , respectively) (Fig. 1). The amide I peak at 1656 cm−1 is regarded as a characteristic for the helical structure of collagen [23], and it is known that cold water fish gelatin undergoes a structural transition from a random coil to a helical conformation at around 15 ◦ C [25]. The spectral changes, therefore, may indicate that FG molecules had an ordered and entangled helical secondary structure at 10 ◦ C. The FG–GA coacervate phase at 10 ◦ C exhibited an amide A peak at a lower wavenumber (3410 cm−1 ) compared to those prepared at higher temperatures (Fig. 1), suggesting that FG–GA coacervate phase had increased hydrogen bonding [24] at a lower phase separation temperature. The stronger hydrogen bonding in FG–GA coacervate phase at a lower phase separation temperature, especially at 10 ◦ C, could be a reason for the enhanced partition of the biopolymers, especially GA molecules, into the coacervate phase with a lower FG:GA value at a lower temperature (Table 1). The FG–GA coacervate phase at 10 ◦ C also showed an amide I peak at 1654 cm−1 (Fig. 1), indicating that the FG molecules in the coacervate phase at 10 ◦ C also had a helical secondary structure.

FG-GA 40 oC 1632

3427

3.3. Steady shear behavior of FG–GA coacervate phase

1523

FG-GA 30 oC 1639

3420

1539

FG-GA 10 oC 1541 1654

3410

4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1) Fig. 1. FTIR spectra of FG, GA, and FG–GA coacervate phases separated at 10, 30, or 40 ◦ C. The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C.

Fig. 2 shows the apparent viscosity (a ) of the FG–GA coacervate phases separated at three different temperatures as a function of shear rate (). ˙ Three corresponding control samples, which are the FG–GA mixtures prepared with the same biopolymer compositions as the coacervate phases, but at pH 8.0, where there is no significant attractive electrostatic interactions between FG and GA molecules, were also investigated. At all the tested temperatures, the coacervate phases showed much higher values of a than the controls, although the biopolymer composition was the same. This may be because the resistance to flow was elevated due to the FG–GA electrostatic interactions in the coacervate phases. Weinbreck et al. [26] also reported that the coacervate phase of whey protein and gum arabic at pH 4.0 had much higher viscosity than the control prepared at pH 7.0 with similar composition.

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Table 2 Carreau model parameters estimated for FG–GA coacervate phasesa separated at three different temperatures and their controls.

FG–GA at 40 ◦ C FG–GA at 30 ◦ C FG–GA at 10 ◦ C Control at 40 ◦ C Control at 30 ◦ C Control at 10 ◦ C

0 (Pa s)

∞ (Pa s)

N

0.250 0.327 1.246 0.042 0.079 0.198

0.046 0.061 0.150 0.016 0.017 0.019

0.36 0.38 0.43 0.22 0.28 0.30

˙ c (s

)

0.0110 0.0380 0.0827 0.0025 0.0028 0.0031

G' (40 oC) G" (40 oC) o

10

2

R

G' (30 C) o

G" (30 C)

0.9973 0.9989 0.9999 0.9992 0.9997 0.9998

G' or G'' (Pa)

Sample

−1

100

G' (10 oC) G" (10 oC)

1

0.1

a

All the phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C. The controls are the FG–GA mixtures prepared at pH 8.0 with the same biopolymer compositions as the coacervate phases, determined in Table 1.

0.01

0.001

All the samples exhibited a shear-thinning flow behavior, and therefore, the well-known Carreau model was employed to describe the viscosity profiles in Fig. 2 [27]. a = ∞ +



0 − ∞

1+

 2 N

(3)

˙ ˙ c

where 0 is the zero-shear viscosity (Pa s), i.e., the limit viscosity at low shear rates, ∞ is the infinite-shear viscosity (Pa s), i.e., the limit viscosity at high shear rates, ˙ c is the critical shear rate (s−1 ) marking the onset of shear-thinning region, and N is a dimensionless exponent related to the slope of shear-thinning region. The Carreau model showed a good fitting to the experimental data with high R2 values (≥0.9973), and the model parameters estimated are listed in Table 2. The values of 0 and ∞ for FG–GA coacervate phase increased by about 5-fold (0.250–1.246 Pa s) and by about 3-fold (0.046–0.150 Pa s), respectively, with decreasing phase separation temperature from 40 to 10 ◦ C (Table 2). This is probably because decreasing the temperature not only reduced the mobility of the biopolymers but also increased the hydrogen bonding between the biopolymers, as observed in the FT-IR analysis. The higher mass fraction of GA, having about 6 times larger molecular weight (382 kDa) compared to FG (58 kDa), at a lower temperature (Table 1) may be also attributed to the increase of 0 and ∞ with the temperature decrease. The controls also showed about 5-fold increase in 0 (0.042–0.198 Pa s) but only a slight increase in ∞ (0.016–0.019 Pa s) with decreasing the temperature. The value of N for FG–GA coacervate phase increased from 0.36 to 0.43 with decreasing phase separation temperature (Table 2), indicating that the coacervate phase was not only more viscous but also more shear-thinning at a lower temperature. The coacervate phase had a higher GA fraction at a lower temperature (Table 1), and it is known that polysaccharide relaxation is mainly responsible for the shear thinning behavior of most concentrated biopolymer mixtures [26]. In addition, the hydrogen bonding became stronger with the temperature decrease, even leading to the formation of helical structure in FG at 10 ◦ C, as observed in the FT-IR analysis. Therefore, the biopolymers in the coacervate phase might undergo more conformational changes and rearrangements at a lower temperature. The controls also showed an increase in N value from 0.22 to 0.30 with the temperature decrease. At any tested temperature, the coacervate phase was found to have a higher N value, indicating greater shear-thinning behavior than the control, although the biopolymer composition was the same. This is probably because the complexes formed by attractive electrostatic interactions between FG and GA molecules, i.e., FG–GA complex coacervates, were subjected to more structural rearrangements than the biopolymers mixed without significant attractive interactions. The value of ˙ c for FG–GA coacervate phase significantly increased by 7.5 times (0.0110–0.0827 s−1 ) with decreasing phase

0.1

1

10

100

ω (rad/s) Fig. 3. Storage (G ) and loss (G ) moduli versus angular frequency (ω) for FG–GA coacervate phases separated at 10, 30, or 40 ◦ C. The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C.

separation temperature (Table 2), implying the increase of Newtonian plateau region at low shear rates. This is quite interesting because the result indicates that at a lower temperature, the coacervate phase was more resistant to the structural deformation at low shear rates, although it showed stronger shear-thinning behavior once the structural deformation and rearrangements occurred above ˙ c . This is probably ascribed to the stronger hydrogen bonding and the reduced molecular mobility at a lower temperature. The controls also exhibited an increase in ˙ c from 0.0025 to 0.0031 s−1 with the temperature decrease, but not as dramatic as the coacervate phase. At any tested temperature, much higher ˙ c value, i.e., wider low-shear Newtonian plateau, was observed for the coacervate phase than for the control. This is probably because at low shear rates, the FG–GA attractive electrostatic interactions might disturb the deformation of GA molecules or other molecular structure, as suggested by Weinbreck et al. [26]. 3.4. Oscillatory shear behavior of FG–GA coacervate phase Fig. 3 shows the frequency sweep of the FG–GA coacervate phases separated at three different temperatures. For the phases separated at 30 and 40 ◦ C, the storage modulus (G ) was found to increase with the angular frequency (ω), but to decrease when the frequency exceeded 50 and 25 rad/s, respectively, implying the occurrence of structural breakdown in the phases due to the reduced intermolecular connectivity at high frequency. However, the G of the coacervate phase at 10 ◦ C continuously increased with the frequency without decreasing, and the coacervate phase obtained at a lower temperature showed a higher G at any frequency. The results confirm the increase of structuration in the coacervate phase with decreasing the temperature, probably due to the reduced molecular mobility, the stronger hydrogen bonding, and the higher GA fraction at a lower temperature, as previously mentioned. The loss modulus (G ) continuously increased with the frequency at all the tested temperatures, and showed larger values than the G values, indicating the liquid-like viscous behavior of the coacervate phases (Fig. 3). Decreasing the temperature also raised the G value, but the increase of G was not as significant as that of G due to the structuration, resulting in the approach of G to G with decreasing the temperature. The coacervate phase at 10 ◦ C even showed a crossover of G and G at a frequency of 73 rad/s, that is, exhibited liquid-like viscous behavior below the crossover frequency and solid-like elastic behavior above it. The

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Fig. 4. CSLM micrographs of FG–GA coacervate phases separated at 40 ◦ C (A), 30 ◦ C (B), and 10 ◦ C (C). The images were obtained from FITC-stained FG molecules. The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C. Bar represents 50 ␮m. (For interpretation of the references to color in text near the reference citation, the reader is referred to the web version of this article.)

FG–GA complexes in the phase at 10 ◦ C might have not enough time to relax to a more favorable structure and dissipate energy above the crossover frequency, and consequently, act more like a rigid gel network, in which the energy imposed by the oscillatory strain could be temporarily stored, leading to the solid-like elastic behavior of the coacervate phase [28]. The coacervate phase of bovine serum albumin and chitosan also showed a crossover frequency, below which G < G (viscous) but above which G > G (elastic), and both dynamic moduli increased with the decrease of temperature, although the temperature was not the phase separation temperature but the temperature of measurements [14]. The coacervate phase of ␤-lactoglobulin and gum arabic also showed a transition from liquid-like to gel-like behavior with a crossover of G and G after an aging treatment for 48 h [29]. The coacervate phase of whey protein and gum arabic also exhibited viscous behavior (G < G ) during the frequency sweep, however, no crossover frequency was reported and the effect of temperature was not investigated [26]. Nevertheless, the coacervate phases of bovine serum albumin-pectin and ␤-lactoglobulin-pectin showed gel-like elastic behavior (G > G ) during the frequency sweep [30,31]. The different oscillatory shear behaviors of the different coacervate phases reflect characteristically distinct structures of protein–polysaccharide complex coacervates.

which were found to be smallest in the coacervate phase at 10 ◦ C, indicating that the coacervate phase at 10 ◦ C had more condensed microstructure than the phase at 30 or 40 ◦ C. A similar CSLM microstructure was also reported by Schmitt et al. [29] for the coacervate phase of ␤-lactoglobulin and gum arabic, which became denser with the loss of water vacuoles upon aging. Fig. 5 shows the SEM micrographs of the three FG–GA coacervate phases. All the coacervate phases displayed a sponge-like porous microstructure due to the entrapment of water molecules within the coacervate structure. The water vacuoles in the coacervate microstructure, i.e., the pores in the micrographs, were observed to become smaller and more homogeneous in size with decreasing the temperature, indicating more compact coacervate microstructure at a lower temperature. According to our observation, therefore, the FG–GA coacervate phase is regarded as a dense and coalesced liquid phase, where FG and GA molecules are homogeneously complexed and networked by electrostatic attractions and noncoulombic interactions, such as hydrogen bonding, to form a continuous matrix in which water vacuoles are dispersed. In the present study, a close relationship was found between the microstructure and the rheological properties of the coacervate phase. The denser the microstructure is, the more viscous, the more shear-thinning, and the more elastic the coacervate phase is.

3.5. Microstructure of FG–GA coacervate phase 3.6. Sol–gel transformation of FG–GA coacervate phase Fig. 4 shows the CSLM micrographs of the FG–GA coacervate phases separated at three different temperatures, where the FG molecules stained with FITC were represented in green. For all the coacervate phases, the green-colored images were similar to the red-colored images from RITC-stained GA molecules (not shown), demonstrating nearly homogeneous distribution of FG and GA molecules in the coacervate phases. The black regions in the images represent water vacuoles inside the coacervate microstructure,

Fig. 6 shows the temperature dependence of G and G of the FG–GA coacervate phases separated at three different temperatures. The temperature was lowered from each phase separation temperature to 3 ◦ C and then raised back to the phase separation temperature at a rate of 0.016 ◦ C/min. During the cooling, all the coacervate phases showed the following notable sequential changes in the moduli; (1) a sudden increase in G immediately

Fig. 5. SEM micrographs of FG–GA coacervate phases separated at 40 ◦ C (A), 30 ◦ C (B), and 10 ◦ C (C). The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C. Bar represents 100 ␮m.

900

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100

(A)

G* = 75.2 ± 1.8 Pa Tg = 3.7 ± 0.1 oC Tm = 6.2 ± 0.1 oC

G' or G" (Pa)

10

Heating

1

0.1

Cooling

G' (Cooling) G" (Cooling) G' (Heating) G" (Heating)

0.01 0

10

20

30

40

o

Temperature ( C) 100

(B)

G* = 79.9 ± 1.8 Pa Tg = 3.8 ± 0.0 oC Tm = 6.6 ± 0.0 oC

G' or G" (Pa)

10

Heating

1 Cooling

0.1

Heating

G' (Cooling) G" (Cooling) G' (Heating) G" (Heating)

0.01 0

5

10

15

20

25

30

Temperature (oC) 100

(C)

G* = 85.7 ± 2.4 Pa Heating

Tm = 6.9 ± 0.1 oC

G' or G" (Pa)

10 Tg = 3.9 ± 0.0 oC

1

Cooling

0.1

G' (Cooling) G" (Cooling) G' (Heating) G" (Heating)

0.01 2

4

6

8

10

o

Temperature ( C) Fig. 6. Changes in storage (G ) and loss (G ) moduli during cooling and heating of FG–GA coacervate phases separated at 40 ◦ C (A), 30 ◦ C (B), and 10 ◦ C (C) at a rate of 0.016 ◦ C/min. The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C. The moduli were monitored at ω = 2 rad/s and  = 1%, and complex modulus (G*) was obtained at 3 ◦ C.

after the cooling started, (2) a rapid increase in G between 6.5 and 7.7 ◦ C, (3) a second sharp increase in G between 5.2 and 5.9 ◦ C, and (4) a crossover of G and G between 3.7 and 3.9 ◦ C. The crossover temperature, at which G exceeded G , was defined as the gelling point (Tg ) of the coacervate phase [32]. The initial sharp increase

of G is probably due to the adjustment of the coacervate system to the cooling conditions. As the cooling proceeds, the mobility of biopolymers is reduced, and the hydrogen bonding between the biopolymers becomes stronger. As discussed in previous sections, these could induce the conformational transition of FG molecules from random coli to helical structure, which was reported to occur at around 15 ◦ C for cold water fish gelatin [25]. The resulting FG helices may participate in the formation of junction zones for thermoreversible gel networks, as known for mammalian gelatins [33]. Consequently, more and stronger inter- and intra-macromolecular connectivities might be formed with the cooling, leading to the remarkable increase, first in viscous fluidity (G ), and then in elastic solidity (G ), followed by the crossover of G and G , i.e., gelation of the coacervate phase. Decreasing the phase separation temperature from 40 to 10 ◦ C was found to slightly increase the gelling point (Tg ) of the coacervate phase from 3.7 to 3.9 ◦ C and also increase the complex modulus (G*) obtained at 3 ◦ C from 75.2 to 85.7 Pa (Fig. 6). This indicates that the coacervate phase obtained at a lower temperature had a higher tendency for gelation and formed a more rigid gel, which may be also attributed to the reduced molecular mobility and the stronger hydrogen bonding in the coacervate phase. In particular, the coacervate phase separated at 10 ◦ C, which is below the random coil-to-helix transition temperature of FG molecules, was found to contain helical FG molecules in our FT-IR analysis, and therefore, the cooling could easily associate the helices for the formation of junction zones, leading to the stronger and faster gelation, compared to the coacervate phases separated at higher temperatures. During the heating of the coacervate phases, the following sequential changes were observed in the moduli; (1) a crossover of G and G between 6.2 and 6.9 ◦ C with sharp decreases in both G and G , (2) a sudden change of G from a steep to a gradual decrease between 9.0 and 10.3 ◦ C, (3) a sudden change of G from a steep to a gradual decrease between 9.8 and 10.6 ◦ C (not observed for the coacervate phase separated at 10 ◦ C). The crossover temperature, at which G went below G , was defined as the melting point (Tm ) of the coacervate phase [32]. On the contrary to the case of cooling, the heating increases the molecular mobility and weakens the hydrogen bonding between the biopolymers. These could loosen the junction zones of gel networks, stabilized by hydrogen bonding, and shift the conformation of FG molecules from an ordered helical to a disordered random coil state. Consequently, the heating might reduce and weaken the inter- and intra-macromolecular connectivities, leading to the melting of the coacervate gel networks and the dramatic losses in both elastic solidity (G ) and viscous fluidity (G ). Decreasing the phase separation temperature from 40 to 10 ◦ C was found to increase the melting point (Tm ) from 6.2 to 6.9 ◦ C (Fig. 6), also indicating that the coacervate phase separated at a lower temperature formed a more rigid and thermo-stable gel. The Tm values were higher than the Tg values for all the three coacervate phases due to the thermal hysteresis, which is a commonly observed indication of reluctance to the thermoreversible sol–gel transformation of a polymeric system. Decreasing the phase separation temperature from 40 to 10 ◦ C increased the difference between gelling and melting points (Tm –Tg ) from 2.5 to 3.0 ◦ C, also indicating the formation of a more ordered and thermo-stable gel by the coacervate phase separated at a lower temperature. It would be also worthwhile to examine the effect of GA, a nongelling biopolymer, on the sol–gel transformation. The temperature dependence of G and G was also examined for the following two controls: (1) an aqueous FG–GA mixture prepared at pH 8.0, where there is no significant FG–GA attractive interactions, with the same biopolymer compositions as the coacervate phase separated at 10 ◦ C (7.7% FG and 7.4% GA, Table 1), (2) an aqueous FG solution prepared at the same concentration as that in the coacervate phase separated at 10 ◦ C (7.7%). The FG–GA mixture prepared

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at 10 ◦ C, having a FG:GA value of 1.0 (Table 1), at which the FG–GA electrostatic interactions at pH 3.55 was reported to be the most intense [3]. 3.7. Viscoelastic behavior of FG–GA coacervate gel Fig. 7 shows the frequency sweep (at  = 1%) of the three FG–GA coacervate gels obtained by cooling the coacervate phases separated at three different temperatures to 3 ◦ C. All the three coacervate gels exhibited a slight increase in storage modulus (G ) and a sharp increase in loss modulus (G ) with increasing angular frequency (ω) (Fig. 7A). The values of G were higher than those of G at any frequency, and the crossover of G and G was not observed. Accordingly, the loss tangent (tan ı = G /G ), an indicative of liquidlike behavior, showed a steep increase with frequency but did not exceed unity (Fig. 7B). The results imply that the three FG–GA coacervate gels can be classified as a weak physical gel formed by noncovalent interactions, such as hydrogen bonding, hydrophobic interactions, and electrostatic interactions [35], having a tendency toward more liquid-like behavior at a higher frequency due to the breakdown of network structure. It was also found that the gel obtained from the coacervate phase separated at a lower temperature had a smaller loss tangent at any frequency (Fig. 7B), also demonstrating that reducing the phase separation temperature can form a more rigid FG–GA coacervate gel network, as previously discussed in the section of sol–gel transformation. 4. Conclusions

Fig. 7. Storage (G ) and loss (G ) moduli (A) and tan ı (B) versus angular frequency (ω) for the gels obtained by cooling the coacervate phases separated at 10, 30, or 40 ◦ C to 3 ◦ C. The coacervate phases were separated from the FG–GA mixture prepared at a total biopolymer concentration of 2% (w/w), FG:GA = 1:1, pH 3.5, and 40 ◦ C.

at pH 8.0 showed a higher gelling point (3.7 ◦ C) than the FG solution (3.1 ◦ C), and also higher G values, indicating that the presence of GA molecules facilitated the gelation of FG molecules. This could be explained that the volume exclusion effect between the two biopolymers, where the free volume of FG molecules is reduced due to their thermodynamic incompatibility with GA molecules, increased the probability of association of FG molecules, and the resulting filler effect of the dispersed GA-rich phase reinforced the FG network and increased the modulus [34]. The FG–GA mixture prepared at pH 8.0 had a gelling point close to that (3.9 ◦ C) of the coacervate phase separated at 10 ◦ C, but much lower G values. Its complex modulus (G*) at 3 ◦ C was measured to be 36.7 Pa, which was only 43% of that (85.7 Pa) of the coacervate phase at 10 ◦ C. The results clearly demonstrated that the presence of GA molecules could significantly reinforce the gel network by their attractive electrostatic interactions with FG molecules, much more than by their filler effect resulting from the thermodynamic incompatibility between the two biopolymers. In the coacervate phase, the GA molecules electrostatically interacted with FG molecules seem to participate in the formation of gel network together with FG molecules to form more compact network structure. The coacervate phase separated at 10 ◦ C showed a higher complex modulus (G*) at 3 ◦ C than the coacervate phases separated at higher temperatures (Fig. 6), although it had a lower FG concentration (Table 1). This may be attributed not only to the existence of FG helices at 10 ◦ C, as previously mentioned, but also to the stronger FG–GA electrostatic interactions in the coacervate phase separated

The present study demonstrated that decreasing the phase separation temperature from 40 to 10 ◦ C significantly influenced the phase volume, composition, steady and oscillatory shear behaviors, microstructure, and sol–gel transformation of FG–GA coacervate phase, as well as the viscoelastic behavior of FG–GA coacervate gel. The decrease in the temperature increased the volume fraction of coacervate phase, and enhanced the partition of the two biopolymers, especially GA molecules, into the coacervate phase. This is probably because the hydrogen bonding, which is known to be majorly responsible for the reversible aggregation of biopolymer complexes during the nucleation-and-growth like process of phase separation, became stronger with decreasing the phase separation temperature, as shown by FT-IR spectroscopy. The coacervate phase became more viscous and more resistant to the structural deformation at low shear rates with decreasing the phase separation temperature, but showed stronger shear-thinning behavior once the structural deformation and rearrangements occurred above ˙ c . The decrease in phase separation temperature significantly increased the G of coacervate phase in the frequency sweep test, and the phase separated at 10 ◦ C even showed a solid-like elastic behavior at a frequency of above 73 rad/s. In addition, the coacervate phase was found to have more condensed microstructure at a lower temperature. The decrease in phase separation temperature increased the values of Tg , Tm , and (Tm –Tg ) but decreased the tan ı of the FG–GA coacervate gels formed at 3 ◦ C, indicating that the coacervate phase separated at a lower temperature had a higher tendency for gelation and formed a more rigid and thermo-stable gel. The major reasons for all the above results may be that decreasing the phase separation temperature reduced the mobility of the biopolymers, increased the mass fraction of GA molecules, strengthened the hydrogen bonding between the biopolymers, and formed FG helical structure at 10 ◦ C, as shown by FT-IR spectroscopy. The FG–GA coacervate gels were classified as a weak physical gel formed by noncovalent interactions, and the presence of GA molecules was found to significantly reinforce the gel network probably due to their attractive electrostatic

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