New insights into protein–polysaccharide complex coacervation: Dynamics, molecular parameters, and applications

For more than a decade, the discovery of liquid–liquid phase separation within living organisms has prompted colloid scientists to understand the connection between coacervate functionality, phase behavior, and dynamics at a multidisciplinary level. Although the protein–polysaccharide was the first system in which the coacervation phenomenon was discovered and is widely used in food systems, the phase state and relaxation dynamics of protein–polysaccharide complex coacervates (PPCC) have rarely been discussed previously. Consequently, this review aims to unravel the relationship between PPCC dynamics, thermodynamics, molecular architecture, applications, and phase states in past studies. Looking ahead, solving the way molecular architecture spreads to macro‐functionality, that is, establishing the relationship between molecular architecture–dynamics–application, will catalyze novel advancements in PPCC research within the field of foods and biomaterials.

a deep understanding of their underlying chemical and macromolecular design principles.
In food science, protein-polysaccharide complex coacervation (PPCC) seems to be a common research topic.These studies have focused on the phenomenological characterization or application of new combinations.In recent years, synthetic polyelectrolytes (PE) and polypeptides with customized sequences have greatly promoted the development of a theoretical understanding of coacervation, especially polymer chemistry-and sequence-controlled coacervation.But in the food industry, natural biopolymers, including proteins and polysaccharides, are still the most important components of complex coacervation considering food regulations and the pursuit of green labels.Therefore, it is valuable to further dig out how the molecular structure and charge properties of proteins and polysaccharides affect complex coacervation.
[22][23][24][25][26] The phase state and relaxation dynamics of PPCC are almost unmentioned topics.In addition, previous reviews mostly discussed the impact of the protein-polysaccharide mixing ratio, pH, and salt concentration on PPCC, and the roles of these factors are highly dependent on the chemical structure of the biopolymers.Therefore, in this review, we hope to unravel from past work the origin of the two phase states of complex coacervates, including liquid coacervates and solid precipitates, and their potential impact on functionality and applications.We anticipate that the phase state and relaxation dynamics are exciting directions for future PPCC research.

Phase states of biopolymer complexes and their relaxation dynamics behaviors
The associative phase separation between proteins and polysaccharides leads to the formation of a biopolymer-rich phase and a biopolymer-poor phase. [27]The biopolymerrich phase can exhibit different phase states, such as liquid coacervates and solid precipitates.When interacting residues on the Ångström scale compete for transient contact on the nanosecond scale, the resulting dynamic multivalent network coordinates relaxation behaviors at different time and size scales, such as submicrosecond-scale exchange of interaction partners, conformation dynamics, and millisecondscale molecular translational diffusion (Figure 1).This coordinated multiscale structural rearrangement causes the biopolymer-rich phase to behave as viscous liquids, which are coacervates.In contrast, when kinetic traps from strong attractive forces limit the multiscale relaxation behaviors, the biopolymer-rich phase in the non-equilibrium state behaves as a solid precipitate (Figure 1).In addition, short-range forces such as hydrogen bonds, hydrophobic forces, and cation-π can also affect the phase state of PPCC because the delicate balance of forces determines whether complexation produces liquid or solid, and these forces are related to the stability of biopolymer structural motifs. [1,28,29]For example, hydrogen bonds may make the conformation of the complex tightly packed and allow inter-and intramolecular hydrogen bonds to replace surface-bound water, thereby creating arrested domains limiting the structural rearrangement and making PPCC behave like a solid. [30,31]This is similar to the physical characteristics of liquids and solids.The components in liquids easily rearrange and change their neighborhood quickly.But in solids, components are caged and do not easily rearrange. [32]Also, chemical reactions tend to occur in liquid environments because reactants in liquids can collide randomly.
However, coacervates are often confused with solid precipitates in food science because these two complexes have similar driving forces and sometimes transform into each other by changing conditions (such as salt concentration and temperature).Moreover, coacervates have also been widely confused with other electrostatic complexes, such as soluble complexes (a complex resulting from insufficient pairable charged groups), cross-linked electrostatic complexes, and electrostatic complex-stabilized emulsions or microcapsules (Figure 2).It is not only a matter of semantics because coacervates have different appearances, structures, and material properties from solid precipitates.Re-examining and elucidating the distinctions and connections of these complexes can benefit their tailored applications.For example, non-equilibrium complexes are the building blocks of layerby-layer assemblies and soluble complexes are used to deliver many active ingredients.Coacervates in biological cells also exhibit different phase and material properties, such as highly fluid and liquid-like droplets or more viscous, viscoelastic, or porous solids or gels, which perform different functions.The phase state of coacervates and its transition within these organisms are often associated with sequence, mutation, and environment.36] Turbidimetry, laser Doppler electrophoresis (ζ-potential), and light scattering are commonly used characterization techniques for PPCC. [38,39]However, these methods cannot provide insight into the phase state of the proteinpolysaccharide complexes.Microscopy is usually used to distinguish between liquid coacervates and solid precipitates.Coacervates appear as droplets with a diameter of 1-50 μm, while solid precipitates exhibit amorphous flocculation (Figure 2).In addition, rheology also provides additional insights.For coacervates, the loss modulus (Gʺ) dominates the linear viscoelastic response.In contrast, the viscoelastic response is dominated by the storage modulus (G′) over the entire frequency range, demonstrating the more solid-like character of precipitates.The point at which a phase transitions (solid to liquid or liquid to solid) can also be identified from frequency sweep results. [40,41]

Phase diagram and composition
In studies of PPCC, the construction of phase diagrams is basically based on turbidity as a function of pH.As the pH changes (from low to high or high to low), pH values corresponding to several important turbidity changes are defined: (1) the pH where the turbidity begins to increase slightly is pH c , which means the formation of soluble complexes and beginning of electrostatic complexation; (2) the pH at which the turbidity rapidly increases is pH φ , implying that soluble complexes assemble into larger insoluble complexes (coacervates or precipitates); and (3) the pH corresponding to maximum turbidity is pH opt , meaning charge neutralization and the maximum yield of complexes (Figure 3A).[44][45] Interestingly, pH c , pH φ , and pH opt changes depend on the charge ratios of biopolymers or charged patches under specific conditions.[47][48][49] In addition, although increasing the temperature lower than the protein denaturation temperature reduces the binding constant of complex coacervation, the effect on pH c , pH φ , and pH opt was limited. [38]This method, however, is limited to the choice of pH c and pH φ , which is highly subjective and is also limited Images of macroscopic precipitate and the microscopic precipitate flocs.Reproduced with permission from Galvanetto et al. [33] and Perry et al. [30] to the preparation of complex coacervates by mixing first and then adjusting the pH, whereas many complex coacervates are prepared by the pH-first method (adjusting proteins and polysaccharides to the same pH and then mixing).In addition, the main components of complex coacervates also include salt and water, in addition to proteins and polysaccharides.The lubrication and relaxation of PE chains by water in the complexes and the doping of salts in ion pairs are closely related to the phase state of the complexes. [50,51]herefore, it is necessary to establish a phase diagram that can represent all components in the coacervate.The thermodynamic phase diagram (binodal curve) also appears in the research of complex coacervation. [39]It takes into account the concentrations and partitioning of biopolymers and salts in the supernatant (left curve) and concentrated phase (right curve) and estimates the water content of coacervates.The two-phase region (coacervate and precipitate) is below the binodal curve, while the single-phase region (where no phase separation occurs) is above the binodal curve.The two phases of each sample are linked by a straight line to represent the distribution of salt in the two phases.Increased salt concentration reduces the polymer concentration of the coacervates through "self-inhibition" and increases the water content.54] The highest point on the binodal curve is the critical salt concentration, that is, the salt concentration that completely inhibits the phase separation (Figure 3C).The protein concentration can be determined by UV-vis and colorimetry, the polysaccharide content can be obtained according to the stoichiometric ratio, and the salt content can be obtained by ionic conductivity; alternatively, the total biopolymer content and salt content can be determined by thermogravimetric analysis.It is important to link various factors to the phase behavior, such as the concentration and distribution of these Micrographs and schematics of (A) liquid-like coacervate droplets, (B) precipitates, (C) cross-linked coacervate particles, and (D) emulsion droplets and microcapsules.Reproduced with permission from Muhoza et al. [37] F I G U R E 3 (A) The change in turbidity of protein-polysaccharide complex coacervates (PPCC) with increased pH.pH c , pH φ , and pH opt indicate the formation of soluble complexes, larger insoluble complexes, and charge-neutral complexes, respectively.(B) Schematic representation of the common phase diagram of PPCC indicating the changes in pH c , pH φ , and pH opt with gradient of factors, such as salt concentration and temperature.(C) Thermodynamic phase diagrams as a function of salt concentration and polymer concentration.The critical point is the highest point of each binodal curve.The dashed tieline defines the salt and polymer concentration for a coexisting coacervate and equilibrium solution.Reproduced with permission from Blocher and Perry. [39]mponents, the critical salt concentration, and the width of the two-phase region.For example, increasing pH from 4 to 6 reduced the polymer concentration in the coacervate and salt resistance by reducing the charge density of the chitosan chains by 22%. [31]Increasing the chain length significantly improved salt resistance because the free energy gain per chain obtained by complexation of long chains far exceeded kT. [55]In addition, lowering the dielectric constant of the solvent (adding organic solvents) increased the polymer concentration and salt resistance by increasing the Bjerrum length and the Coulomb energy of electrostatic interactions. [56]However, the relevant phase behavior results are all obtained from PE-PE complex systems, and there are few relative studies on PPCC.

THERMODYNAMICS OF PPCC
After the introduction of the term "complex coacervation" by Bungenberg de Jong and Kruyt, [4] Overbeek and Voorn proposed the earliest theories of complex coacerva-tion, suggesting that coacervation arises from a combination of electrostatic attraction and mixing entropy of polyions and their counterions based on Flory-Huggins. [57]Currently, it is generally accepted that electrostatic interactions are not always the main driver for some systems (especially PE-PE complex coacervation).The entropy gain from counterion release can overcome the enthalpy contribution of electrostatic interactions.But for the more intricate PPCC, the thermodynamic results show a remarkable diversity, although it is generally accepted that the entropy increase caused by the release of counterions and water molecules in protein-polysaccharide complex coacervation is limited, because almost all proteins and polysaccharides (except sulfated and phosphated polysaccharides) are weak PEs, and globular proteins lack flexible chains.There are four types of thermodynamic parameters in the existing PPCC studies (Table 1): (1) Most protein-polysaccharide combinations exhibit an exothermic interaction (ΔH < 0) and reduced entropy (ΔS < 0), that is, only exothermic dominated complex TA B L E 1 Four typical thermodynamic results of protein-polysaccharide complex coacervates obtained by isothermal titration calorimetry.

Underlying mechanism
Refs.
Exothermic interaction dominated complex coacervation (ΔH < 0, ΔS < 0) The reduced mobility and flexibility of the biopolymers after association may, that is, vibrational and conformational entropy decrease.
(3) Some protein-polysaccharide combinations show exothermic interactions and an increased entropy behavior; that is, they both drive complex coacervation despite of different relative magnitudes.The driving force from the exothermic interaction of ovalbumin (OVA)-pectin and zein-chitosan (pH 4) combinations exceeds that of the entropy increase. [59,75]In contrast, the contribution of increased entropy for the α-lactalbumin-chitosan, [76] BSA-κ-carrageenan, [76] and zein-chitosan (pH 3) combinations is greater than that for exothermic interactions. [59]However, it should be noted that using mass concentrations in the isothermal titration calorimetry (ITC) fitting model may misjudge the relative magnitudes of ΔH and TΔS.(4) The ITC titration curves of some protein-polysaccharide combinations show double inflection points, such as BLG-acacia gum, [77] chickpea protein-Persian gum, [78] and BLG-pectin. [79,80]Considering the nonspecific nature of electrostatic interactions, these studies used the "two sets of sites" model to describe the processes of electrostatic complexation and coacervation, rather than two different kinds of independent binding sites.Electrostatic complexation is mainly driven by exothermic interactions (exotheric + decreased entropy or |ΔH| > |TΔS|), while coacervation is dominated by entropy gain or exothermic/entropy gain (entropy gain was close to exothermic).
][71][72] This showed that the charge strength of the PE is not the only decisive factor.It should be emphasized that it is difficult to understand whether these diverse thermodynamic parameters arise from differences in the structural (molecular weight [Mw], chain length, chain flexibility) or charging characteristics (charge density, charge pattern, charge anisotropy) of proteins and polysaccharides or from different solvent conditions for complex coacervation by comparing fragmented studies.This still requires systematic comparative studies that control the variables.After all, on the basis of the entropy-enthalpy balance, there is also a balance between enthalpy changes of various interactions and a balance between different types of entropy changes during complex coacervation (Figure 4).Furthermore, although dynamics dominate the phase state of the electrostatic complexes, the differences in complexation strength and solvent exclusion during the formation of coacervates and solid precipitates are bound to affect thermodynamic parameters.Therefore, it is interesting to establish the relationship between the thermodynamic parameters and the phase state.

Rheological strategies
Complex coacervation is a dynamic process determined by the molecular architecture of biopolymers and environmental conditions.When these factors are considered, understanding the dynamics of complex coacervates becomes complicated.Rheology is suitable quantitative method for understanding the dynamics of PPCC.The dynamic relaxation-related measurements are usually performed on complex coacervates that have been equilibrated for several days.The dynamic response of complex coacervate on various time scales can be obtained by rheological analysis of the equilibrated coacervates.Small-amplitude oscillatory shear detects the linear viscoelastic response of complex coacervates and provides information about their viscosity, modulus, and relaxation behavior. [40,41,81]The viscosity of proteinpolysaccharide electrostatic complexes is much higher than that of polysaccharides, proteins, and protein-polysaccharide F I G U R E 5 Rearrangement and reversibility of coacervate structure.(A) The viscosity of whey protein-gum arabic complex coacervates with liquid character completely recovered during shear rate reduction, adapted with permission from Weinbreck et al. [85] (B) The viscosity of the canola protein isolategum arabic precipitate was not observed to recover during the test period, adapted with permission from Stone et al. [86] (C) Fluorescence recovery after photobleaching (FRAP) experiments of single stranded DNA-poly(l-lysine) (ssDNA-pLL) complex coacervate droplets with fluorescently labeled ssDNA, adapted with permission from Bos et al. [87] After the fluorescence in the middle part of the coacervate droplet is bleached by the laser beam, it gradually recovers due to the reversibility of the coacervate structure.
mixtures without electrostatic complexation.Hence, the electrostatic complexation greatly increases the viscosity of the mixed systems.The increased viscosity originates from the "adhesion" between interacting charged groups, which reduces the fluidity of the system.This is similar to the proposed rheology model based on "sticky" Rouse/Zimm dynamics for PE. [40,41,82]Shear thinning is observed in protein-polysaccharide electrostatic complexes.Stronger electrostatic interaction leads to a more significant shear thinning. [83,84]Interestingly, due to the structural deformation caused by high shear forces, the flow curve of coacervates at decreasing shear rate exhibits a pronounced hysteresis compared to the flow curve at an increasing shear rate.The viscosity was fully recovered at low shear rates for whey protein isolate (WPI)-gum arabic coacervates with typical liquid characteristics (Gʺ was three to seven times higher than G′) during the shear rate decrease (Figure 5A). [85]The full recovery of WPI-chitosan electrostatic complexes with liquid characteristics in the low-frequency region (crossover of G′ and Gʺ) costs more than 2 h. [83]In contrast, for canola protein isolate-gum arabic electrostatic complexes with solid characteristics (G′ > Gʺ), no viscosity recovery was observed during the test (Figure 5B). [86]requency sweep data provide response characteristics of complex coacervates over different time scales.][90][91][92][93][94][95][96] In contrast, the liquid-like or viscous behavior (Gʺ > G′) dominates in some other PPCC combinations. [85,97]The crossover of Gʺ and G′ curves also occurs in some PPCC combinations.(D-F) The relaxation time spectrum of PDMAEMA-PAA coacervates with various KCl concentrations (labeled on the right of the curves).From left to right, these three figures are stress relaxation modulus, relaxation time spectra, and rescaled relaxation time spectra with scaling factors τ c shown in the inset.Reproduced with permission from Spruijt et al. [40] An increased salt concentration shifts the crossover of the two moduli to high frequencies, that is, the longest relaxation time (the reciprocal of the frequency corresponding to the crossover point) decreases. [83]But for the combination of gelatin A-zedo gum, gelatin A-cress seed gum, gelatin Bzedo gum, and gelatin B-cress seed gum, G′ dominated in the low frequency (<1 Hz) and high frequency (>10 Hz), while Gʺ dominated in the mid-frequency region (1-10 Hz).Unfortunately, most studies of PPCC have not been accompanied by microscopy observations, so it is hard to clearly confirm the correlation of the liquid-like behavior obtained from frequency sweep results (Gʺ > G′, tan δ > 1) with the liquid droplets observed by microscopy.But, gelatinκ-carrageenan complex coacervates fit this hypothesis. [97]oreover, in the studies of PE and heteroprotein complex coacervates, the phase state of the electrostatic complexes obtained by frequency sweep measurements was consistent with that obtained by microscopy. [28,81,98,99]The frequency sweep results even provided preliminary information on saltor temperature-induced phase state transitions. [98,100]he viscoelastic response of a single coacervate sample can only obtain information on a narrow time scale.The dynamics information on a wider time scale (a few decades) can be obtained by superimposing the viscoelastic response curves of a series of factor gradient samples (Figure 6A-D).This method has been used to understand the relaxation behavior of synthetic PE electrostatic complexes, natural PE electrostatic complexes, and protein-PE electrostatic complexes. [41,56,82,101]Time-salt superposition is the most common strategy because salts accelerate rearrangements between paired charged groups by weakening the interactions between charged groups.Using an arbitrary curve or the crossover of G′ and Gʺ as a reference, the frequency (ω scaled = ωτ c ) and/or the modulus (G′ scaled = G′/G c and Gʺ scaled = Gʺ/G c ) are rescaled using shift factors (τ c and G c ) so that they overlap the main curve.Briefly, τ c is used to interpret the dependence of the apparent relaxation time on the salt concentration, whereas G c is used to understand the dependence of the polymer concentration on the salt concentration.In addition, time-temperature superposition, time-pH superposition, and time-alcohol superposition have been used to deeply understand the influencing mechanism of various factors on the dynamics and relaxation behavior of complex coacervates. [56,102]Even multiple superpositions, such as time-temperature-salt superpositions, time-salt-alcohol superpositions, and time-temperature-water superpositions, have also been used to understand complex synergistic mechanisms. [56,98,103]However, the superposition strategy requires that all viscoelastic response curves have a similar shape.This may mean that not all combinations of oppositely charged biopolymers are applicable.The superposed results can be checked by the smoothness of the resulting superposed tan δ curves or graphs of the Cole-Cole plot (Gʺ scaled vs. G′ scaled ).
The determination of the relaxation time spectrum is also one of the rheological methods to observe the relaxation behavior of complex coacervates.It can be obtained from converting frequency sweep results and step-strain experiments. [40,81]Briefly, the relaxation time spectrum can detect the entire range of dynamic timescales (including a terminal relaxation time) for a given coacervate from milliseconds (frequency sweep) to hours (step-strain experiment) (Figure 6E-G).This may indicate molecular relaxation models at various time scales, such as the relaxation of a small part of the polymer chain in a PE complex coacervate in a short time scale, followed by extensive rearrangement of ionic bonds (rouse-like stress relaxation) in an intermediate time scale, and the relaxation of the entire polymer chain after the terminal relaxation time. [40]Once the relaxation spectra of coacervates with gradient factors have a similar shape, a superposition can be performed on the relaxation time spectra to understand how these factors relate to the relaxation behavior of coacervates at different timescales.Rheological strategies were highlighted because they provide powerful tools for studying the dynamics of complex coacervates.More details and introductions can be found in our citations and a series of excellent reviews. [27,40,41,104,105]

Fluorescence-based strategies
[108] The exchange and molecular transport rate of the labeled biopolymers inside or between the inside and outside of the coacervate is measured by recording the fluorescence recovery curve of the area bleached by the laser beam and fitting the mathematical model. [38]Combining FRAP results with chemical structure and formation conditions helps to understand the dependence of "fluidity" on each factor. [106]However, although FRAP is considered a golden method for detecting the fluidity and diffusion of coacervates in many disciplines, it is difficult to understand the dynamic behavior at the molecular scale inside the coacervates. [33,109]n complex coacervation systems, Förster resonance energy transfer (FRET) occurs under dipole-dipole interactions when the emission spectrum of the photon donor (tryptophan or fluorescent dye) partially overlaps with the absorption spectrum of the acceptor (binding molecule or fluorescent dye) and the donor and acceptor are close enough (<10 Å).The donor resonantly transfers energy to the acceptor with high efficiency.The emission fluorescence of the donor chromophore weakens or disappears, and the fluorescence of the acceptor chromophore is mainly emitted.The energy conversion efficiency (E) between the donor and the acceptor is inversely proportional to the sixth power of the spatial distance between them (r = R 0 [ 1 E − 1] −6 , where r and R 0 are the interaction distance and Förster radius, respectively).Therefore, E is very sensitive to changes in spatial position and can reflect the distance between fluorescent donor and acceptor.Based on this principle, the ratio of the fluorescence intensity of the acceptor to the donor or the change in E over time is used to monitor the exchange dynamics of the donor and the acceptor between the coacervates when a coacervate containing a donor and a coacervate containing an acceptor exist in the same system. [110]For example, take the complex system with only donor and only acceptor as the zero point (A/D min , 0% FRET), take the donor and acceptor 1:1 as the maximum point (A/D max , 100% FRET), and use dimensionless parameter α A/D to evaluate the exchange rate at time t ( A∕D = A∕D t −A∕D min A∕D max −A∕D min ). [111]In some cases, the increase in FRET efficiency depends not only on the exchange rate but also on factors such as initial fluorophore number, fluorophore size, Forster radius, and mixing ratio. [112]urthermore, when the donor and acceptor are close to each other, the FRET transfer from donor to acceptor is faster, and the lifetime of the fluorophore becomes shorter.Therefore, combining FRET with fluorescence lifetime imaging (FLIM) can also estimate the average donor-acceptor distance and molecular density changes within the coacervate by measuring the lifetime of the donor fluorophore.FLIM can also detect microscopic viscosity changes and grid size changes of complex coacervates through the fluorescence lifetime of the molecular rotor (sulfonated boron-dipyrromethene) because the fluorescence lifetime depends on its intramolecular rotation rate. [87]Recently, Galvanetto et al. probed these long-range chain remodeling times τ r in single-molecule FRET experiments combined with nanosecond fluorescence correlation spectroscopy to study the timescales of their conformational interconversions. [33]luctuations in the interdye distance lead to fluctuations in donor and acceptor emission intensities, which can be quantified by correlating the fluorescence signals.In summary, fluorescence-based technology provides a way to understand the dynamics of complex coacervates at the molecular scale, but it still needs to be combined with nuclear magnetic resonance, small angle X-ray scattering, and molecular dynamics simulation to fully peek into multiscale relaxation behavior fully.A detailed explanation of these techniques can be found in our previous review. [38]

Charge density of polysaccharides
Considering the charge anisotropy and complexity of proteins, current studies mainly focus on the relationship between polysaccharide charge density and PPCC.One compared the complex coacervation between a protein and different weak/strong polysaccharides, such as carboxylated and sulfated polysaccharides.The other one compared the complex coacervation between a protein and the same polysaccharide with different charge densities, resulting from the modification of the polysaccharides, such as esterification of pectin (degree of methylesterification), carboxymethylation of carboxymethyl cellulose (CMC) (degree of substitution), and sulfation of carrageenan (the number of sulfate groups).Generally, a greater charge density of the polysaccharides leads to broader conditions for complex formation and associative phase separation.In a comparison of six carboxylated (xanthan gum, sodium alginate, and gum arabic) and sulfated (chondroitin sulfate, dextran sulfate, and λ-carrageenan) polysaccharides, a higher charge density of the polysaccharides corresponded to higher pH c and pH φ of PPCC. [113]The same is true for the same polysaccharide with different charge densities.A decreased methyl esterification degree of pectin results in an increase in pH c and pH φ1 associated with pea protein isolate-pectin complex formation, while a decrease in pH φ2 was observed, associated with complex dissociation. [58]λ-Carrageenan with three sulfate groups has the highest pH c and pH φ1 when complexed with WPI, compared to κand ι-carrageenans, which contain one and two sulfate groups per disaccharide repeating unit, respectively. [114]Moreover, a stronger charge density deepens the associative phase separation on the wrong side (pH > pI) caused by protein charge anisotropy.Polysaccharides with higher charge density also enable protein-polysaccharide complexes with critical salt concentrations. [114,115]The breadth of PPCC conditions varies by the polysaccharide charge density, which means that the complex sensitivity to pH and salt can be modulated to achieve environmental response/controlled release or protection of sensitive components.For example, the αamylase in the α-amylase-pectin complexes (linear charge density of pectin was 6.17 C/m) is almost completely inactive after only 40 min at pH 3.But the α-amylase activity of αamylase-λ-carrageenan complexes (linear charge density of λ-carrageenan was 61.2 C/m) only decreases slowly and still retains 40% of enzyme activity after 120 min. [116]The high charge density of λ-carrageenan improved the resistance of α-amylase to adverse environments through stronger electrostatic interactions.It should be noted that both complexes are amorphous precipitates.Regarding the assembly structure, a high charge density of polysaccharides increases the binding constant of protein-polysaccharide complexation, causing a more compact structure. [66,115]The pectin with higher charge density even reduced the size of protein domains and inhibited protein self-aggregation. [117]Notably, it remains to be seen whether a high charge density of polysaccharides increases the size of the complexes.The OVA-CMC (with a degree of substitution of 0.7) complex is significantly larger than the OVA-CMC (degree of substitution of 1.2) complex. [118]The turbidity of WPI-κ-carrageenan is also greater than that of WPI-λ-carrageenan and WPI-ι-carrageenan. [114]lthough it is generally believed that reducing the overall charge density promotes the formation of liquid coacervates rather than solid precipitates. [28,119]For some protein-polysaccharide complexes, simply reducing the overall charge density of the polysaccharide is not sufficient to cause coacervate formation. [120]PPCC is also affected by the local charge density of the polysaccharide, especially when the overall charge density of the polysaccharide is small.For example, the random (low local charge density) or blockwise (high local charge density) distribution of methyl-esters gov-erns the local charge density of pectin.Coacervate droplets are only formed when both the overall and local charge densities of the pectin are low (Figure 7). [115,121]When the overall charge density of pectin is low, randomly distributed local charges may favor the dynamic rearrangement of ion pairs, with a weakened exotherm and increased entropy gain.Conversely, localized charges of the blockwise distribution increase the exotherm and weaken the entropy change. [115]

Protein charge anisotropy and charge distribution
Although proteins are often regarded as uniformly charged spheres in PPCC studies, charged residues are usually asymmetrically distributed on the protein surface, which results in charge anisotropy.It seems reasonable to propose that hydrated protein surfaces mediate the electrostatic interactions between proteins and other biopolymers since such intermolecular attractions are on the order of kT. [122]In PPCC studies, one of the best examples of protein charge anisotropy is that protein-polysaccharide interactions often begin at pH c (the pH at which the turbidity begins to increase) where the protein and PE are of like charge, pH c < pI for polyanions or pH c > pI for polycations, which are termed binding on the "wrong side" of pI.
For BSA and BLG with similar pI, the negatively charged patch on the BLG surface enables it to bind PDADMAC with high affinity (low pH c ) at higher ionic strengths, which makes BLG in the BSA-BLG mixed protein system preferentially bind to PDADMAC when the ionic strength is higher than 50 mM, thereby achieving protein separation.Similarly, the BLG-A isoform in BLG has a stronger negatively charged patch than the BLG-B isoform due to the substitution of glycine at position 64 by aspartic acid, which makes BLG-A in BLG at 100 mM ionic strength preferentially bind to PDADMAC to achieve selective coacervation. [123]enetic engineering was used by Kapelner and Obermeyer to precisely control the number and location of charges in green fluorescent protein (GFP). [124]The less charged isotropic mutant iso-GFPs (-7 and -12) and the less charged anisotropic mutant tag-GFPs (-7 and -12) can both interact with the strong polycation poly(4-vinyl N-methyl pyridinium iodide) (qP4VP) and form coacervates.But the highly charged iso-GFPs (-18 and -24) and qP4VP formed precipitates through liquid-solid phase separation due to kinetic trapping, whereas highly charged tag-GFPs (-18 and -24) formed coacervates with qP4VP (Figure 8A).Recently, protein surface charge patches were parameterized further to quantify the effect of protein charge anisotropy on coacervation.Four mutants of superfolded green fluorescent protein (sfGFP, -1.5 and -1.8) whose charge distribution on the surface varied from near-perfectly isotropic to near-perfectly anisotropic were synthesized.The mutant with medium patches was found to strongly phase-separate and form solid precipitates with qP4VP at 0 and 50 mM NaCl.The mutants with large and extra-large patches formed complex coacervates with qP4VP at 0 and 50 mM NaCl (Figure 8B). [125]his difference has been attributed to the large negative patches likely allowing for greater conformational freedom of the interacting chain or chains, leading to liquid complex coacervates, whereas smaller patches do not allow the confor- The relationship between overall and local charge density of pectin and the morphology and phase state of pectin-lysozyme complexes.(A) A high charge density of low methoxylated pectin causes the pectin-lysozyme complex to appear as an amorphous precipitate.(B) Although an increase in the degree of methoxylation reduces the charge density of pectin, the high local charge brought about by blockwise distribution makes the pectin-lysozyme complex still present an amorphous precipitate.(C) On the basis that the increase the degree of methoxylation reduces the charge density of pectin, the low local charge caused by the random distribution of methyl galacturonate makes the pectin-lysozyme complex appear as coacervate droplets.The confocal laser scanning microscopy (CLSM) images were adapted with permission from Antonov et al. [115,121] F I G U R E 8 (A) Electrostatic surface potential of isotropic iso-green fluorescent protein (GFP) and tag-conjugated anisotropic tag-GFP (blue for positive and red for negative) and their phase states after electrostatic complexation with poly(4-vinyl N-methyl pyridinium iodide).Adapted with permission from Kapelner and Obermeyer. [124](B) Charge patches of GFP mutants (blue for positive and red for negative) and their phase states after electrostatic complexation with poly(4-vinyl N-methyl pyridinium iodide).Adapted with permission from Kim et al. [125] mational freedom, leading to solid complexes.Notably, this differs from the previously effect of polysaccharide local charge density on the phase state of the complexes.
In addition, the protein charge anisotropy also affects its uptake in ternary coacervates.For example, when proteins are encapsulated by poly(L-lysine trifluoroacetate) and poly(D,L-glutamate sodium salt), lysozyme with clustered charged patches on the surface significantly increases its uptake in the coacervate and increases the sensitivity of the system to modulation by other parameters, which is different from BSA and human hemoglobin, where the surface charges are uniformly dispersed on the surface. [126]

Chain length (molecular weight) of biopolymers
Compared to charge density, far too little attention has been paid to the effect of biopolymer chain length or Mw on PPCC, although they may significantly affect PPCC.For example, chitosan with Mw of 303 kDa was degraded into chitosan with the same charge density but smaller Mw by ultrasonication.The reduced Mw of chitosan increased the turbidity of the BLG-chitosan complexes, which was attributed to an increase in the total number of particles and the size of the complexes.Interestingly, the decreased Mw of chitosan did not alter the charge stoichiometry of the BLGchitosan complexes, but reduced their reaction exotherm. [127]his was different from peptide-peptide and polysaccharidepolysaccharide complex coacervation, in which the decreased chain length reduced the formation of PE complexes and the critical salt concentration of the coacervate-solution boundary. [128]A reduced chain length of HA also reduced the formation range of HA-chitosan complexes and inhibited complex coacervation because biopolymers with shorter chains lost more configurational entropy as they moved from the diluted to the concentrated phase and had lower entropy gains from counterions and water expulsion. [129]Therefore, the discrepancy might be because PPCCs were mostly driven by electrostatic interactions rather than increasing entropy.
The chain length also affects the phase state and relaxation behavior of PPCCs.The pH range of liquid coacervate formation for the low-Mw gelatin-sodium alginate combination was much broader than that of the high-Mw gelatin-sodium alginate combination. [130]The intersection between G′ and Gʺ moves to the same higher frequency with decreasing polymer chain length, suggesting that a decreasing polymer chain length may accelerate dynamic relaxation. [81]

Chain stiffness (flexibility) of biopolymers
A good consensus has been achieved on the effect of chain stiffness (flexibility) of biopolymers on complex coacervation.The stiffness (flexibility) of the chains is closely related to the persistence length (l p ), which is the sum of the electrostatic persistence length (l e ) and the bare persistence length (l 0 p ). [24] A chain is more flexible when its contour length is longer than its persistence length, so a longer permanence length generally reduces the flexibility. [131]A high charge density and low ionic strength increase the electrostatic per-sistence length because the electrostatic repulsion between the charged chain segments increases the bending energy. [24][134][135][136] As a result of increased chain flexibility (decreased l p ), a greater number of segments touched the sphere, and the chain undergoes a structural transition and collapse at the surface of the sphere.In contrast, stiff chains (increased l p ) make less contact with proteins. [136]Briefly, stiff chains have less binding degrees of freedom and generally weaker binding forces, and thus have lower critical salt concentrations than flexible chains.
The experimental results are consistent with simulations.The chain flexibility of PDADMAC (l 0 p = 2.5 nm) is higher than that of HA (l 0 p = 4.2 nm).BSA and BLG interact with PDADMAC with a higher affinity than with HA.The binding affinity of BSA-PDADMAC is twofold higher than that of BSA-HA, but for BLG-PDADMAC, it is ninefold higher than that for BLG-HA.The chain flexibility has a greater impact on the BLG, which may be related to the fact that smaller protein BLG lacks a positive domain with low curvature. [74]In addition, the higher flexibility of PDAD-MAC enables greater exenthalpic ion pairing, but it has a lower TΔS because PE configurational entropy is lost as PEprotein complexes form.In contrast, semi-flexible PE chains such as HA and chitosan (l 0 p = 6.5 nm) suffer less loss of configurational entropy when forming complexes. [129]APPLICATIONS

Enzyme immobilization and protein encapsulation
Encapsulating proteins by complex coacervates is believed to be bioinspired because it mimics the protein concentration within the cell and applies crowding effects to stabilize proteins and enzymes, thus preserving their folded structure and bioactivity. [137]Compared to other carriers encapsulating proteins and enzymes, complex coacervates have the advantages of a pure water environment, simple preparation, high encapsulation efficiency, environmental response, good biocompatibility, and concentration effect. [138]Numerous studies have used different kinds of complex coacervates to encapsulate or extract proteins and enzymes, such as PPCCs, complex coacervate core micelles, and core vesicles formed by diblock copolymers, polypeptide coacervates, PE coacervates, and heteroprotein coacervates. [139,140]This review mainly focuses on protein encapsulation based on PPCC.
Since complex coacervates are isolated from water, the enzymes can be easily separated from the reactant for reuse.The polysaccharides can also increase the enzyme resistance to adverse pH conditions and high temperatures.For example, immobilized lactase is commonly used in the food industry to produce lactose-free milk.Although the electrostatic complexation with carrageenan or alginate slightly reduces the accessibility to the substrate and enzymatic activity of lactase, complexation with especially carrageenan, which has a stronger affinity for lactase, greatly improves the stability of lactase in acidic pH (<5) and at high temperatures.Moreover, the catalytic efficiency of the electrostatically complexed and dissociated lactase is similar to that of the native enzyme. [141,142]Although it has been widely reported F I G U R E 9 (A) The poly(acrylic acid)-polyethyleneimine complexes in the form of coacervate protected enzymatic activity of α-amylase at low pH for a long time, whereas the complexes in the form of precipitate were difficult to protect α-amylase.Reproduced with permission from Kübelbeck et al. [144] (B) The ability of β-conglycinin-lysozyme coacervates to protect curcumin from chemical degradation in aqueous environments was significantly ahead of precipitates.Reproduced with permission from Zheng et al. [145] (C) Protein-polysaccharide solid precipitates capture the probiotics in a loose gel network, and the probiotics are interspersed between the network structures.Reproduced with permission from Bosnea et al. and Mao et al. [146,147] In contrast, liquid coacervates can trap probiotic cells in tight spherical droplets.Reproduced with permission from Bosnea et al. and Silva et al. [148,149] that electrostatic complexation can unfold the enzyme structure, thereby possibly reducing the enzyme activity, the enzyme activity usually recovers once it dissociates from complex coacervates. [142]Furthermore, although polysaccharides with higher charge densities lead to more unfolding of the enzyme structure upon complexation, polysaccharides with higher charge densities also confer more protection on the enzymes.For example, compared to the α-amylase-pectin combination, λ-carrageenan-α-amylase complex coacervates with stronger affinity form over a wide pH range.The λcarrageenan-α-amylase combination can effectively protect the enzyme activity as a soluble complex with smaller particle size even in formation conditions far away from complex coacervation. [116]The complex coacervation based on three macromolecules can effectively avoid the protein unfolding during electrostatic complexation because the coacervates are mainly constructed by two PEs, and the protein is only doped into it as a guest molecule. [126,137,143]As an example, the enzymatic activity of α-amylase is not reduced when it is encapsulated by poly(acrylic acid)-polyethylenimine complexes, and the complexes in the form of coacervates protect α-amylase from the adverse effects of low pH for several hours, whereas the complexes that exist as precipitates do not protect the α-amylase (Figure 9A). [144]It is unclear whether there is also a difference in the stabilization behavior of coacervates and precipitates toward enzymes in the protein-polysaccharide binary complex coacervation system.In addition, careful selection of the polysaccharide species and modification properties are required for a specific enzyme geometry and charged patches to encapsulate and dissociate enzymes under target conditions, thereby reducing adverse effects on enzyme activity.
In addition to enzyme immobilization, PPCC has also been used to modulate the enzymatic activity of polysaccharide hydrolases, [150,151] recover target proteins and enzymes, [152] purify proteins by selective coacervation, [113,153] mask the unfavorable taste of plant proteins, [154] and target the delivery of protein drugs and digestive enzymes. [155,156]verall, there are still many pending questions about protein encapsulation based on PPCC.For example, (1) how the chemical structure and charge characteristics of proteins and polysaccharides modulate the response of complex coacervates to the environment for the protection and targeted delivery of functional proteins; (2) in the complex coacervation system based on three macromolecules, how the chemical structures and charge characteristics of proteins and polysaccharides affect the doping of proteins; and (3) comparing the multiscale structure, dynamics and rheological properties of coacervates and precipitates after encapsulating proteins to understand why these two complexes behave differently when encapsulating and stabilizing proteins.

Encapsulation of bioactive
The first practical application of complex coacervation was using a combination of gelatin and gum arabic to microencapsulate dyes to manufacture carbonless copy paper. [157,158]ince then, numerous studies have used complex coacervation to encapsulate bioactive ingredients with various physicochemical properties using different combinations of biopolymers.Complex coacervation, as one of the classic microencapsulation technologies, can protect bioactive ingredients from light, oxygen, heat or other extreme conditions, thereby improving their stability and maintaining bioactivity, which is not only based on the phase barrier, but also the multivalent interactions and concentration effect that regulate the reaction conditions and equilibrium of chemical degradation. [159]Generally, the ingredient that is used as a guest and the preparation method determine the type of complex coacervates (core-shell or network) rather than the type of biopolymer, although the structure and physicochemical properties of the biopolymer affect the phase state of the complex coacervates, their encapsulation capacity, and guest stability.[162][163][164][165][166] The phase state of these complexes is related to the structure and properties of the biopolymers.Some researchers have proposed that the stability of bioactive molecules in liquid coacervates may be significantly better than that of amorphous solid precipitates because solid precipitates do not have micron-scale spherical domains (Figure 9B). [145,167]Interestingly, in cells, the transition of membraneless organelles from the liquid to solid state is also associated with a loss of functionality.However, the relationship between the phase state of complex condensates and the stability of bioactive molecules still needs more confirmation.It should be emphasized that generally complex coacervates encapsulating bioactive components need to be dried for storage and transportation, but the concentration effect of liquid coacervates may enable the application of "liquid capsules" encapsulating cargo and produced by liquid-liquid phase separation. [168,169]][172][173] First, the protein and oil are mixed and homogenized into an O/W emulsion, polysaccharide is added into the emulsion and the system is homogenized again.Next, its pH is adjusted to make the protein and polysaccharide complex at the oil-water interface.A small part of research also directly uses protein-polysaccharide electrostatic complexes to construct emulsions. [174,175]After macro-phase separation, the "complex coacervates" are collected and dried to obtain microcapsules, which usually exhibit a shell-core structure.But these studies usually only provide the surface morphology obtained by scanning elec-tron microscopy, and it is difficult to distinguish the internal structure.Therefore, the co-localization of the host and guest molecules in the microcapsules will help to understand the internal structure of the microcapsules to analyze the stability mechanism of bioactive ingredients.In addition, the relationship between the dynamics of complex coacervates and their structure at the oil-water interface also needs to be further elucidated.

Encapsulation of cells
Ingestion of sufficient living probiotic cells (≥10 6 CFU/g) confers health benefits to the host.However, these living cells are lost during food processing and storage and in extreme environmental conditions (high temperature, acidic pH).Therefore, some studies have tried to reduce this loss by microencapsulating probiotic cells through complex coacervation.Protein-polysaccharide solid precipitates capture the probiotics in a loose gel network, and the probiotics are interspersed between the network structures (Figure 9C). [146]or example, WPI-κ-carrageenan precipitates exhibiting a network structure significantly enhance the survival of Lactobacillus plantarum after exposure to gastric juice and bile salts. [176]In addition, the spherical particles formed by spray-drying sodium caseinate-sugar beet pectin and pea protein isolate-sugar beet pectin precipitates have a better cell (Lactobacillus rhamnosus GG) stability than the lyophilized sponge network structure during digestion. [177][180] WPI-gum arabic coacervates can provide better acid resistance (pH 2) to probiotics (Lactobacillus paracasei subsp.paracasei and Lactobacillus paraplantarum) than precipitates formed by heating WPI and gum arabic. [147]Furthermore, WPIgum arabic coacervates allow the exchange of metabolites and nutrients within it with the bulk liquid medium by their dynamic exchange nature, thus enabling these probiotic cells to grow and metabolize nutrients within the coacervate structure. [180,148]

Biomaterials for electrospinning
Generally, complex coacervates are well separated from water and have good biocompatibility, low interfacial tension, environmentally responsive material properties and functionality, and thus are great potential biomaterials. [183,184]lectrospinning can be used to produce high surface area polymer fiber mats in the diameter range 10 nm-5 μm, which can be used in wound healing, water remediation, catalysis, and food packaging applications. [185]Most watersoluble polymers are charged PE.During electrospinning, the concentration of charged polymer must be higher than the critical entanglement concentration, otherwise the polymer solution does not have enough stability to maintain continuous fibers. [185]However, the repulsive interaction between the charged groups in the polymer backbone causes F I G U R E 1 0 (A) Smooth continuous fibers were electrospun from the coacervates containing 1.50-1.70M KBr.Within this range, the diameter of fiber decreased with increasing salt Below this range, the coacervates were too viscous to extrude through the spinneret, while above this range, the coacervate was too thin to be collected for further processing.Reproduced with permission from Meng et al. [186] (B) Increasing the molecular weight (Mw) of hyaluronic acid (HA) sharply slowed down the dynamics of HA-chitosan complex coacervates, which led to the excessive gelation of coacervate ink for 3D printed cell scaffolds and the broken structure during extrusion, whereas too fast dynamics caused the coacervate cannot be maintained shape after extrusion due to shape relaxation.Reproduced with permission from Khoonkari et al. [190] (C) Polyacrylic acid-isoprenyl oxy poly(ethylene glycol)/tannic acid coacervate, which has good adhesion and self-healing capabilities, allowed the skin wound of Sprague-Dawley rats (8 weeks old) to recover well within 14 days.Reproduced with permission from Wang et al. [191] (D) The thickness of coacervate films obtained by all drying methods (anneal, air-drying, and freeze-drying) and spin rates (1000-3000 rpm) decreased with increasing salt concentration because the viscosity and dynamics of the coacervate decreased with increasing salt concentration.Reproduced with permission from Kurtz et al. [192] the viscosity of the charged polymer aqueous solution to sharply increase with the polymer concentration, which hinders the electrospinning process.In addition, toxic solvents and cross-linking agents are often required during the spinning process, which may cause cytotoxicity and hinder the integration of water-soluble polymer-based fiber mats into advanced materials.[188] Molecular interactions provide jet cohesion instead of entanglements.Interestingly, the coacervate fibers formed by spinning have good encapsulation capabilities of cargo molecules and can even prevent them from dissolving in water. [187]In addition, complex coacervation even enables the electrospinning of oligomers (1 < chain length N anion /N cation < 6/9), as it can avoid the need for entan-glement, eliminating the requirement of polymer chain length and entanglement for electrospinning. [189]he relaxation behavior and phase state of complex coacervates are crucial to their processability during electrospinning.The presence of a small amount of ethanol (3 wt%) can accelerate the evaporation of the solvent and enhance the cohesion of the coacervate and decrease the dielectric coefficient of the solvent, thereby obtaining nanofibers of 100-200 nm.However, when the ethanol concentration is increased to 8 wt%, the slowed relaxation and sharply increased viscosity significantly raised the fiber diameter to ∼10 μm. [188]Similarly, smooth, continuous fibers could only be electrospun from poly(4-styrenesulfonic acid, sodium salt)-poly(diallyldimethylammonium chloride) (PSS-PDADMAC) coacervates formed between 1.50 and 1.70 M KBr; the coacervates containing less than 1.50 M KBr were too sticky to be extruded through the spinneret, whereas the coacervates containing more than 1.70 M KBr were too thin to be collected for further processing (Figure 10A). [186]n the future, fully revealing the connection between molecu-lar properties, relaxation behavior, and fiber quality will help fabricate advanced biofibrous materials by electrospinning.

Biomaterials for cell scaffolds
Recently, coacervates have been used to construct cell scaffolds to their biocompatibility, insolubility, underwater adhesion, injectability, and encapsulating capability.For example, the PPCCs formed by electrostatic interactions between mussel adhesion protein (MAP) and HA were used to retain transplanted stem cells in defective articular cartilage in situ.A higher Mw of HA significantly increased the viscosity and G′/Gʺ of MAP-HA coacervates, slowing down the dynamics of coacervates and allowing better maintenance of MAP-HA in cartilage defects and preventing dispersion into the joint cavity.The original storage modulus of MAP-HA coacervate was almost maintained (∼1407 ± 3.66 Pa) in water for 7 days without swelling.Therefore, it retained and prolonged the survival of transplanted stem cells on chondral defects and thereby better regenerate the damaged articular cartilage compared with the application of fibrin glue. [193]Similarly, in another study using HA-chitosan as ink for 3D printed cell scaffolds, increasing the Mw of HA and the density of additional intermolecular H-bonds also sharply slowed down the dynamics of complex coacervates.Too slow dynamics (relaxation time ≈ 10 s) led to excessive gelation of coacervate ink, and the structure was broken during extrusion; too fast dynamics (relaxation time ≈ 0.01 s) caused the coacervate cannot be maintained shape after extrusion due to shape relaxation (Figure 10B).Therefore, besides shear-thinning behavior and fast self-healing, maintaining the relaxation dynamics of coacervates within a reasonable range is the key to making coacervate materials that both possess acceptable plasticity and structural rigidity. [190]

Biomaterials for wound repair
Bioadhesives made from polymers can promote wound closure and tissue repair and are expected to be ideal replacements for sutures.Polymeric materials are the most commonly used substrates for wound dressings, but a humid environment or exposure to water can reduce adhesion.The nature of the liquid-liquid phase separation makes complex coacervates help to overcome this problem.For example, gelatin-sodium alginate complex coacervates encapsulating epidermal growth factor and fibroblast growth factor, respectively, can be used to promote wound healing in diabetic mice and reduce pro-inflammatory cytokines. [155,194]Similarly, HA-lysozyme complex coacervation significantly promotes wound healing in a full-thickness skin defect model, and its efficient self-healing properties help to overcome breakage or deformation. [195]Recently, coacervates based on tannic acid with pyrogallol groups, inspired by the underwater adhesion of sandcastle worms and mussels, were considered to be enhanced bioadhesives.Dynamic hydrogen bonds and covalent cross-links provide strong adhesion to achieve adhesion, self-healing after fracture, promoting wound closure and healing, and protecting wounds from bacteria and physical trauma (Figure 10C). [191,196]In the forthcoming years, elucidating the intricate associations between coacer-vate dynamics and the consequential impacts of antibacterial and growth factors is poised to advance and enhance the wound healing aptitude of coacervate-based interventions.

Biomaterials for films
PPCC has also been used to make edible and degradable films.Films constructed from chitosan-WPI and gelatin-Persian gum complex coacervates have good mechanical properties. [197,198]In contrast, although the mechanical properties of the film formed by the electrically neutral soy protein isolate (SPI)-pectin complex are better than those of 5% SPI alone, the difference in tensile strength (MPa) between complex films with different mixing ratios was not significant. [199]Similarly, the mechanical properties of the films formed by the neutral gelatin-CMC complexes are lower than those of the composite films formed at other mixing ratios. [200]Moreover, the mechanical strength of low methoxyl pectin and sodium caseinate films in charge-neutral precipitates was lower than that of sodium caseinate alone and other non-stoichiometric complexes. [201,202]However, related studies of films constructed by synthetic PE complex coacervates seem to shed light on the reasons for the above seemingly contradictory results.205] In contrast, solid synthetic PE complexes are poorly plastic due to strong electrostatic interactions, but salts can be used to plasticize PE complexes into liquid coacervates to prepare thin films. [192]Therefore, the influence of the dynamics and phase state of the PPCC on the film cannot be ignored.

CONCLUSION AND OUTLOOK
PPCC provides an effective strategy for encapsulating bioactive food ingredients within aqueous matrices, with a consistent focus on investigating the formation conditions and encapsulation properties.However, the phase state and dynamics of coacervates are still an emerging field.Therefore, this review discussed the basic characteristics and discrepancy of the two phase states of protein-polysaccharide associative phase separation products, namely, liquid coacervates and solid precipitates, and attempts to explore the connection between thermodynamics, dynamics, molecular architecture, function, and phase state from sporadic clues at this stage.Looking ahead, the reliance of PPCC functions and applications on its phase state and dynamics will drive us to delve deeper into the mechanism and consequence behind the phase state, such as (1) the relationship between the molecular structure of biomacromolecules and the dynamics, thermodynamics, composition, and multiscale structure of PPCC; (2) the interplay between the thermodynamics and dynamics governing PPCC; (3) how the dynamics and multiscale structure of PPCC influence its functionality and diverse applications; (4) investigating biopolymer combinations and establishing a property library of various coacervate combinations for the development of high-performance coacervate materials; (5) integrating complex coacervation with advanced processing techniques, such as microfluidics, to precisely engineer advanced nano-and microscale materi-als, including nanoparticles, microreactors, biosensors, and drug delivery vehicles; and (6) furthermore, in various fields such as polymers and artificial cells, strategies involving comb-like polyethylene (comb PE), [206] rigid polysaccharides such dextran, [207] and yeast cell fragments [208] have been employed to create steric repulsions around coacervates, thereby ensuring the long-term dispersion of coacervate droplets.It is imperative to explore analogous strategies to prevent macroscopic phase separation in PPCC, as this can potentially pave the way for the development of novel functionalities inspired by biological systems.

F I G U R E 1
Schematic diagram of relaxation behaviors of liquid coacervates and solid precipitates at different time and size scales.(A) In coacervates, dynamic multivalent networks of competing contacts among residues exchange in nanoseconds (intact ionic bonds shown as green solid lines, broken ionic bonds as green dotted lines).(B) At scales higher than the network correlation length, the rapid exchange between interaction partners on the submicrosecond timescale passes to the chain reconfiguration.(C) The translational diffusion of biopolymers inside coacervates.(D) Images of macroscopic coacervate phase and the microscopic coacervate droplets.(E) Strong attractive forces between residues make contacts irreversible through kinetic traps.(F) At higher scales, it is difficult for interacting partners to exchange, limiting changes in conformational dynamics.(G) Therefore, submicron diffusion dynamics are limited.(H)

F I G U R E 4
Complex coacervation is affected by both enthalpy (ΔH) and entropy changes (ΔS).The total entropy change and total enthalpy change of the system are obtained by summating multiple types of entropy changes and interactions, respectively.Left panel: events related to entropy change, and right panel: interactions related to enthalpy change.

F
I G U R E 6 (A-C) Time-salt superposition analysis of poly(N,N-dimethylaminoethyl methacrylate)-poly(acrylic acid)(PDMAEMA-PAA) coacervates with various KCl concentrations.From left to right, these three figures are frequency sweep data, rescaled time-salt superposition of frequency sweep data, and shift factors τ c for time-salt superposition.Reproduced with permission from Liu et al.