Interactions and phase behaviors in mixed solutions of κ-carrageenan and myofibrillar protein extracted from Alaska Pollock surimi

Accepted Manuscript Interactions and phase behaviors in mixed solutions of κcarrageenan and myofibrillar protein extracted from Alaska Pollock surimi

Tao Zhang, Xiaoqi Xu, Zhaojie Li, Yuming Wang, Yong Xue, Changhu Xue PII: DOI: Reference:

S0963-9969(17)30850-5 doi:10.1016/j.foodres.2017.11.080 FRIN 7202

To appear in:

Food Research International

Received date: Revised date: Accepted date:

15 July 2017 27 November 2017 30 November 2017

Please cite this article as: Tao Zhang, Xiaoqi Xu, Zhaojie Li, Yuming Wang, Yong Xue, Changhu Xue , Interactions and phase behaviors in mixed solutions of κ-carrageenan and myofibrillar protein extracted from Alaska Pollock surimi. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi:10.1016/j.foodres.2017.11.080

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ACCEPTED MANUSCRIPT Interactions and phase behaviors in mixed solutions of κ-carrageenan and myofibrillar protein extracted from Alaska Pollock surimi

Tao Zhang, Xiaoqi Xu, Zhaojie Li, Yuming Wang, Yong Xue*，Changhu Xue* Department of Food Science and Engineering, Ocean University of China, Qingdao 266003, PR China * Corresponding author. Tel.: +86-0532-82032597; Fax: +86-0532-82032468.

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E-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract

In the present work, we provide insight into electrostatic interactions and phase behaviors in mixtures of myofibrillar protein (MP) and κ-carrageenan (KC) of various pHs (8.0–3.0) and biopolymer weight ratios [R, from 1:1 to 20:1; total concentration = 0.05% (w/w)] through turbidimetric analysis, dynamic light scattering (DLS) and

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zeta potential analysis, and optical microscopy. At R = 1: 1, critical pH values (i.e., pHc, pHΦ1, and pHΦ2), which indicate phase transitions interrelated to the formation of

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soluble or insoluble MP-KC complexes, were observed at pH 7.6, 6.8, and 3.6,

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respectively. As the ratio increased, the pHmax shifted from 4.9 to 5.8. A similar trend was observed for the isoelectric point of MP-KC mixtures, as determined by zeta

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potential measurements. The maximum interaction indicated by the highest turbidity occurred at pHmax = 5.3, at a ratio of 5:1, whereas pHc remained constant during

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acidification. The changes in electrostatic interactions and transformations of phase behaviors accompanying the complex formation and disassociation processes were

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further supported by particle size distribution analysis and optical microscopic

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observations of MP-KC mixtures (R = 1:1) at different pH values. This work fills the previous lack of studies on phase behaviors of surimi protein and colloidal polysaccharide in liquid system, and lay the foundation to provide a new way to solve

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problems of interactions between protein and polysaccharide during the processing of surimi products.

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Key words: myofibrillar protein, κ-carrageenan, electrostatic interactions, phase behaviors

1. Introduction 2

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Proteins and polysaccharides are natural food hydrocolloids that are extensively applied as functional ingredients in the food industry. The physicochemical and functional properties of these compounds play important roles in several processed foods, including enhancing emulsibility and foamability, influencing texture and struc

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tural stability, and facilitating the delivery of bioactive ingredients to targeted sites (Singh, Bohidar, & Bandyopadhyay, 2007; Schmitt & Turgeon, 2011). These two

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types of biopolymers are frequently included together in many kinds of food systems, as complexes formed between proteins and polysaccharides function better than either

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of these biopolymers alone (Guzey, Kim, & McClements, 2004). Therefore, providing insight into interactions between proteins and polysaccharides is necessary for

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optimizing the physical and chemical performance of food products, improving textural properties, and developing novel foods such as fabricated foods.

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Proteins and polysaccharides coexisting in a system can display one of the following three behaviors: co-solubility, thermodynamic incompatibility, and complex

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formation (de Kruif & Tuinier, 2001). Most biopolymers mixed in solutions are

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thermodynamically incompatible, resulting in segregative phase separation owing to different chemical natures. In contrast, in very dilute solutions, proteins and polysaccharides are co-soluble and stable owing to mixing driven by entropy (De

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Kruif & Tuinier, 2001; Turgeon et al., 2003). Segregative and associative phase behaviors occur with increasing biopolymer concentrations, as entropy decreases

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under these conditions. Segregative phase behavior emerges when two biopolymers are incompatible (Elmer et al., 2011); when the proteins and polysaccharides carry similar net charges, they repel one another, leading to each biopolymer-rich phase separated (Braudo, Plashchina, & Schwenke, 2001). Conversely, associative phase separation occurs when biopolymers with opposite electric charges electrostatically attract one another, dividing the system into biopolymer-rich and solvent-rich domains (De Kruif, Weinbreck, & de Vries, 2004). Interactions between proteins and polysaccharides influence biopolymer solubility, viscoelastic gel formation, and interface performance (Tolstoguzov, 3

ACCEPTED MANUSCRIPT Damodaran, & Paraf, 1997). Recently, phase separation of protein-polysaccharide mixing systems to alter the physical and chemical properties of foods has attracted considerable attentions (Çakir & Foegeding, 2011; Chung, Degner, & McClements, 2013; Rohart & Michon, 2014). Previously, Turgeon et al. (2003) reported that controlling the phase behaviors of hydrocolloids and proteins could facilitate the

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improvement of the quality of gelled foods and in the development of new food structures. More recently, Van den Berg et al. (2009) demonstrated that the phase

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separation commonly employed in restructuring food is effective for controlling and generating the structural elements of foods at various partical scales. Moreover, the

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complicated interplay between the phase-separation and gelation processes might also be useful for controlling the microscopic network structure (Jin et al., 2015). Our

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previous work (Zhang, et al., 2017) on phase behaviors involved in surimi gel system found that phase separation of myofibrillar protein and kappa-carrageenan was

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enhanced as the polysaccharide ratio and temperature increased, while the gel networks were significantly influenced by the κ-carrageenan concentration.

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Numerous studies have focused on characterizing interactions between proteins

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and polysaccharides in solution over the last 10 years, especially concentrating on the liquid state and utilizing solutions or emulsions (Jones & McClements, 2011). To date, most studies of protein-polysaccharide mixing systems have examined the

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interactions and phase behaviors of polysaccharides with plant proteins, such as soy protein isolates and pea protein isolates, and animal proteins, including whey proteins,

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sodium caseinate, milk proteins, egg white protein, gelatin, β-lactoglobulin, and bovine serum albumin (Schmitt, & Turgeon, 2011). However, there seems to be little research on the phase behaviors of mixtures containing muscle-derived proteins. One of the most common representatives of this food type is surimi products. Previous reports have identified multiple hydrocolloids that are thought to improve the sensory, textural, and functional properties of restructured fish gels, typically, surimi, providing a new channel for designing novel surimi or restructured fish products (Martín-Sánchez et al., 2009; Ramírez et al., 2011). Myofibrillar proteins (MP, also called “salt-soluble proteins”) are the most 4

ACCEPTED MANUSCRIPT important components in determining the mechanical and functional properties of surimi food products. MP has manifested the ability to produce 3-dimensional network structures and viscoelastic gel matrices, which contribute to the texture and sensory perception, such as hardness, chewiness, and adhesiveness, of foods (Khoury, 2004). Heating treatment forces MP to expose hydrophobic groups, aggregate, and form an intertwined network structure (Alting, 2003).

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κ-Carrageenan (KC) extracted from red algae is a linear and sulfated

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polysaccharide, with repeating units of galactose and 3, 6-anhydrogalactose (Trius & Sebranek, 1996). KC has been utilized in a broad range of applications in the food

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industry for its thickening, water-holding capacity and gelling properties (Verbeken et al., 2005). Notably, KC can improve the texture and sensory properties of gels formed

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by salt-soluble meat proteins (SSMP) (DeFreitas et al., 1997). On one hand, KC can form an independent network that supports and stabilizes the reticulated framework of

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gelled proteins by filling interstitial spaces; on the other, interactions between SSMP and KC could increase the viscoelasticity of gels.

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In the present research, we investigated the interactions and phase behaviors of

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MP and KC in aqueous solutions. Specifically, through turbidity analysis and zeta (ζ) potential measurements, we evaluated the effects of pH and biopolymer mixing ratios on the formation of soluble or insoluble complexes and the alteration of phase

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association or separation. Additionally, dynamic light scattering (DLS) and optical microscopy analyses were applied to describe changes in particle size in solution and

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to establish phase diagrams and schematic pictures. These results can help to explain the blending system of surimi protein and commonly used colloidal polysaccharides, and provide solutions to the problem of protein and polysaccharide phase behaviors in the processing of surimi products.

2. Materials and methods

2.1. Materials 5

ACCEPTED MANUSCRIPT Frozen Alaska Pollock surimi (grade AAA) was purchased from Jincan Foods Co., Ltd. (Qingdao, China) and maintained at -20°C until use. KC was purchased from Yantai Sheli Hydrocolloids Co., Ltd. (Haiyang, China). All chemicals used were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd.

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(Shanghai, China).

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2.2. Preparation of MP and KC stock solutions and mixtures

A sample of surimi (at 4°C for 3 h) was thawed and added to five volumes of

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Tris-maleate buffer (50 mM KCl and 20 mM Tris-maleate; pH 7.0) containing 3 mM NaN3 to protect against bacterial growth. The surimi paste was then homogenized and

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centrifuged at 8,000 × g using an Anke GL-20G-II centrifuge (Anting Scientific Instrument Factory, Shanghai, China) at 4°C for 10 min. The resulting precipitate was

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washed twice with Tris-maleate buffer (0.6 M KCl and 20 mM Tris-maleate; pH 7.0), homogenized, and centrifuged at 10,000 × g at 4°C for 2 min, incubated at 4°C for 30

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min to allow for protein dissolution, and centrifuged again at 8,000 × g at 4°C for 20

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min. The supernatant was then isolated and precipitated by mixing with 10 volumes of distilled water (KCl diluted to 0.06 M), and the resulting precipitate (MP) was dissolved in 0.6 M KCl for use. The protein content of the MP sample was measured

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as 40.21 ± 0.43 mg/mL via the Biuret method (Gornall et al., 1949). The purification of MP was measured by SDS-PAGE with results attached in the Supplementary

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material, demonstrating the sample is pure enough for the following experiments. Raw KC powder was dispersed in distilled water containing 3 mM NaN3 and stirred for 2 h at 45°C. The solution was dialyzed against distilled water at pH 7.0 to eliminate excess salt; the water was changed several times throughout the 24 h dialysis period. Insoluble particles were then removed by injecting the final solution through a cellulose acetate membrane with a pore size of 0.45 μm (Merck, Darmstadt, Germany). Finally, pure KC powder was obtained by vacuum freeze-drying. Stock solutions (0.05%) were prepared by mixing 0.05 g of pure KC powder or MP in 100 g of distilled water, stirring at 4°C for 2 h, and incubation without stirring 6

ACCEPTED MANUSCRIPT for 24 h at 4°C. These stock solutions were then utilized to generate the panel of biopolymer mixtures with different ratios [R = MP: KC (w/w), 1:1 to 20:1, corresponding to R1, R2, R3, R4, and R5, respectively]. Solutions were maintained at a constant total concentration of 0.05% biopolymer (w/w). The pH values of the solutions were adjusted between 3.0 and 8.0 using NaOH and HCl solutions. Prepared

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mixtures were maintained at 4°C until use.

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2.3. Turbidimetric analysis

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Changes in the turbidity of solutions containing individual biopolymers or biopolymer mixtures during acidification from pH 8.0 to pH 3.0 were measured using

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a Unico-7200 spectrophotometer (Unico Co., Shanghai, PR China) at 600 nm and at room temperature (25°C ± 1). Ultrapure water was used to set transmittance to 100%

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for calibration. Each sample was measured in triplicate every 2 min, and mean values were used for analysis. According to the concept of Weinbreck et al. (2003), the

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critical pH points pHc, pHΦ1, and pHΦ2 were used to indicate structural transitions

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during acid titration, were slight increases in turbidity, and the intersection between maximum turbidity (pHmax) and two curve tangents, respectively.

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2.4. Particle size measurement

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The influence of pH on particle size distribution in solutions containing MP or KC alone, or a 1:1 mixture of MP and KC [constant concentration: 0.05% (w/w)] were determined by measuring the diffusion coefficients (D, m2/s) of these solutions via dynamic light scattering (DLS). These experiments were carried out using a Malvern Zetasizer Nano ZS90 device (Malvern Instrument, Worcestershire, UK) equipped with a He–Ne laser at a wavelength of 633 nm and at 25°C. Samples were evaluated in triplicate to allow for statistical analysis.

2.5.Zeta (ζ)-potential analysis 7

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Similar to particle size measurement, the effects of biopolymer mixing ratio and pH on the ζ potentials (mV) of the mixed systems as well as the corresponding homogenous MP and KC solutions (0.05%, w/w) were determined by measuring the electrophoretic mobility of each solution using a Malvern Zetasizer Nano ZS90

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(Malvern Instrument, Worcestershire, UK) at 25°C in triplicate.

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2.6.Optical microscopy

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MP and KC complexed mixtures were microscopically characterized using an Olympus BX41 optical microscope (Olympus Optical Co., Tokyo, Japan) equipped

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with a digital camera (model DP Controller; Olympus Optical Co., Tokyo, Japan) controlled by a DP Manager version 3.1 image processor. For these analyses, 50 µL of

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each sample was placed on glass slides and observed at 40 magnification.

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3. Results and Discussion

3.1.Effects of pH on changes in turbidity Fluctuations in turbidity are generally considered to result from the formation

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and dissociation of protein polysaccharide complexes, as the turbidity varies primarily because of changes in the mass and size of the system (Xia, Dubin, & Dautzenberg,

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1993). The effect of pH (8.0–3.0) on MP-KC complex formation [R = 1:1, total biopolymer concentration = 0.05% (w/w)] was investigated as shown in Figure 1. During acid titration of the MP-KC mixture, we observed four distinct changes in turbidity values: pHc, pHΦ1, pHmax, and pHΦ2. Of these, the turbidity transition points pHΦ1, pHmax, and pHΦ2 were defined by the intersections of the two tangents, as introduced in the Methods section. Likewise, we observed changes in the turbidity of the control solutions containing MP or KC alone during the acidification process; however, the turbidity of these solutions, due to conformational changes and aggregation of proteins/polysaccharides induced by acidification, was much lower 8

ACCEPTED MANUSCRIPT than that of the MP-KC mixtures. MP and KC mixtures were transparent, indicating co-solubility, at pH values higher than pHc (7.6). Meanwhile, there was a slight increase in the turbidity of these solutions at pH 7.6, suggesting the formation and growth of soluble complexes between MP and KC. Notably, while the pHc value was greater than the isoelectric

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point (pI) for MP (5.97), as both MP and KC are negatively charged, indicating that repulsive coulombic forces should have prevented the formation of complexes,

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studies by Weinbreck et al. (2003), Sperber et al. (2009), and Klassen, Elmer, & Nickerson (2011) demonstrated that non-covalent attractions between polysaccharides

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and proteins contribute to the formation of soluble complexes at pH values higher than the pI of the protein. Thus, the initial formation of soluble complexes at pHc was

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probably due to interactions between reactive groups on the MP and sulfate groups on KC molecules as well as hydrogen bond and other intermolecular interactions.

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Upon further acidification to pHΦ1 (6.7), the MP-KC mixture underwent a shift from transparent to turbid, based on elevated turbidity measurements. This turbidity,

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which increased sharply, corresponded to the formation of insoluble MP-KC

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complexes, implying the aggregation of smaller complexes and leading to the onset of macroscopic phase separation. Subsequently, at pH 4.9 (pHmax), the turbidity of the solution rose to 0.864, indicating the maximum formation of insoluble complexes

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between MP and KC. After continued acidification to pHΦ2 (pH 3.6), insoluble complexes formed and began to agglomerate and become sedimentary, as decreasing

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pH values result in the exposure of greater numbers of reactive sites within polysaccharide chains, thereby facilitating further interactions with proteins (Niu et al., 2014). Thus, the turbidity values of the solution decreased as the coacervates precipitated at the bottom of the tube. As shown in Figure 5, the zeta potential of KC was negative within a pH range of 8.0 to 3.0, while the zeta potential of MP was positive when the pH dropped below the pI. As such, MP and KC formed aggregates under these conditions via intermolecular positive and negative charge-charge attractions. Insoluble complexes began to disassociate and became co-soluble biopolymers at 9

ACCEPTED MANUSCRIPT pHΦ2, which was due to progressive protonation of the reactive groups of the polysaccharides and proteins, resulting in both MP and KC becoming positively charged. Our results were different from the pH-dependent phase transitions pertaining to the formation of β-lactoglobulin-pectin, bovine serum albumin-pectin and ovalbumin-gum arabic complexes described by Sperber et al. (2009), Li et al.

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(2012), and Niu et al. (2014), respectively. In these previous studies, insoluble complexes began to dissolve in solution and form co-soluble biopolymers without any

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aggregation.

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3.2. Effect of biopolymer mixing ratio (R) on turbidity changes

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The mixing ratio (R) of proteins and polysaccharides affects the charge balance in a system, which subsequently alters complex formation (Turgeon, Schmitt, &

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Sanchez, 2007). Figure 2 shows the effects of changes in R (from 1:1 to 20:1) on complex formation between MP and KC at a constant concentration (0.05%, w/w).

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Within a pH range of 8.0 to 3.0, turbidity changed significantly as the proportion of

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MP increased. Specifically, as R increased, critical pH values (pHΦ1, pHmax, and pHΦ2) shifted towards higher pHs (Figure 3). The highest turbidity observed for a protein: polysaccharide ratio of 5:1 was at pHmax = 5.3, which indicated the highest level of

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MP-KC complex formation and that the majority of the MP molecules would be available to reach a charge balance (electro-neutrality) for the insoluble complex. We

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inferred that nearly all charged groups on the KC chains combined with proteins, and that proteins achieved a saturated state after R reached a certain level. As such, at higher R values, more MP molecules would be available to bind to KC chains. Similarly, Wang et al. (2007) observed an optimal ratio of 20:1 while increasing the ratio of β-lactoglobulin:pectin from 5:1 to 40:1. However, the turbidity of the complexes was reduced when R increased from 5:1 to 20:1. With the excess of protein, protein molecules had to compete with each other to interact with polysaccharides, leading to polysaccharide chain stretching and a higher degree of MP dispersion, and consequently, to a reduced degree of aggregation and turbidity. However, there was no 10

ACCEPTED MANUSCRIPT significant change in pHc upon alteration of R (Fig. 3). Consistent with these finding, Girard et al. (2004) previously reported that pHc remained constant despite fluctuations in the mixing ratio of proteins and polysaccharides in a β-lactoglobulin-pectin system. Likewise, Vinayahan et al. (2010) observed a similar phenomenon for mixtures of bovine serum albumin and gum arabic, and suggested

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protein, which occurred independent of the mixing ratio.

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that soluble complexes consisted of single polysaccharides and a certain amount of

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3.3.Particle size measurement

To better understand the impact of pH on the formation and disassociation of

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MP-KC complexes and to provide further insights into interactions between these two biopolymers, the particle size (z-average) was plotted as a function of pH for mixtures

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monitored by DLS (Niu et al., 2015). Meanwhile, homogenous MP and KC solutions (pH 7) were utilized as controls. These two solutions at other pHs were also measured

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but not showed because of no significant difference, and Figure 1 has displayed the

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turbidity of these two solutions changed slightly with various pHs, indicating that the particle size of themselves experienced inconspicuous fluctuations. The biopolymer particles showed a significant difference over various pHs (from

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8.0 to 3.0), as shown in Figure 4. When homogenous MP and KC solutions were mixed together, the particle size increased sharply during acid titration. At pH 8, the

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z-average size was 283.8 nm, which was not markedly different from that of the MP and KC solutions, indicating that both biopolymers were co-soluble and underwent a slight degree of complex formation, as the absence of substantial complex formation was due to the negative charge of both biopolymers. At pH 7, the peak intensity of the z-average size increased from 38.2% to 41.4%, and the particle size was more centralized than that observed at pH 8. With further acid titration, there was a significant increase in particle size to 1043.4 nm and then 2240.6 nm at pH 6 and 5, respectively. Based on the above results, we concluded that insoluble complexes formed and grew as the pH decreased. This conclusion is supported by the fact that 11

ACCEPTED MANUSCRIPT MP would become positively charged and KC negatively charged at lower pH values (Fig. 5), resulting in a relatively strong electrostatic attractive interaction between MP and KC. Moreover, a two-phase system developed, wherein one phase was biopolymer-rich and the other was solvent-rich, and the particle size of the MP-KC system reached a maximum value.

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Interestingly, the particle size began to decrease upon acidification to pH < 5. This effect was likely due to conformational changes in MP and KC at lower pH

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values, which exposed a greater number of binding sites, resulting in intermolecular interactions between positive residues on MP and negative sulfate groups on KC and

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facilitating the formation of insoluble complexes and the gradual production of coacervates. There was a fairly centralized distribution in particle size at pH 3, and

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particles were smaller in value compared with those observed at pH 4, where the solution exhibited two distinct particles (Figure 4): the signal on the left in Figure 4

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likely corresponded to MP-KC complexes while that on the right part likely corresponded to coacervates. During acidification, insoluble complexes began to

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dissociate due to progressive protonation of the anionic groups on the KC structure

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and the denaturation and unfolding of MP (Da Silva et al., 2006). Increasing the degree of acidification would likely break the equilibrium between formation and dissociation of complexes, and disintegrate insoluble complexes into soluble

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complexes or even into non-interacting protein molecules and polysaccharide chains

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(Dickinson, 2008).

3.4.ζ-potential analysis

ζ potential is commonly applied to determine surface charge density, providing important information regarding the formation and stability of a complex as well as interactions between charged proteins and polysaccharides (Soleimanpour et al., 2013). As mentioned above, stable complex formation between MP and KC occurs primarily through electrostatic interactions between oppositely charged biopolymers. Thus, we examined the electro-kinetic potential difference (ζ,) for homogenous MP 12

ACCEPTED MANUSCRIPT and KC solutions and for MP-KC mixtures [1:1 to 20:1 (w/w)] during acid titration from pH 8.0 to pH 3.0 (Figure 5). The ζ potential of homogenous MP (0.05%, w/w) solutions increased from 21.92 mV to 33.53 mV during titration from pH 8.0 to 3.0, due to protonation of the MP amine (-NH2) and carboxyl groups (-COOH). In addition, the isoelectric point of MP, where the ζ value was 0, was pH = 5.97. Similarly, the ζ potential of homogenous

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KC solution increased from 49.25 mV to 22.11 mV, due to the decreased ionization

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of sulfate residues on the KC molecular chain, during acidification. While MP molecules were positively charged only when the pH was below its pI, KC molecules

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were negatively charged over the entire pH range, suggesting that complex coacervate formation occurred between the MP-KC biopolymers due to electrostatic interactions.

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The ζ potential of the MP-KC biopolymer mixture reflected the sum of ζ-potential values of the MP-KC complexes and non-interacting individual

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biopolymers in solution. Overall, ζ potential increased as the ratio increased from 1:1 to 20:1 within the pH range tested. At a 1:1 ratio, the presence of KC shifted the pI of

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MP from 5.97 to nearly 3.0. Furthermore, this R1 mixture exhibited an electrophoretic

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mobility similar to that of the KC solution, as the ζ potential remained negatively charged throughout the pH range. As the mixing ratio (i.e., protein levels) increased, these values shifted towards higher pH values, reaching a pI of 5.48 at a MP-KC ratio

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of 20:1 MP-KC ratio, which was close to that of MP alone. This observation indicated that complexes formed between MP and KC were stabilized by electrostatic

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interactions. The effect of pH on the ζ-potentials for ovalbumin, pectin, and their complexes were studied previously (Jones & McClements, 2010). Similar to our findings, ovalbumin-pectin complexes had intermediate ζ-potentials and the pI increased from 3.55 to 4.55, depending on elevated ovalbumin:pectin ratios. These data indicated that the formation of these complexes occurred largely via electrostatic interactions between the anionic carboxyl groups of pectin and the cationic amino groups of ovalbumin.

3.5.Optical microscopy and schematic diagram 13

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The morphology and surface structure of the complex droplets were observed using an optical microscope (Figure 6). Only a few visible protein aggregation particles were found at pH 8, confirming the low turbidity of the system under the initial conditions (Figure 1). As the pH decreased to 7, the visible particles became

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larger, indicative of soluble complexes, and further acidification to pH 6 significantly increased the extent of this insoluble complex formation. At pH 5, the complexes

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began to aggregate to produce coacervates, resulting in the clumped status of the particles (Figure 6D), as the turbidity of the system plateaued (Figure 1). Continuous

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acid titration led to dissociation of the complexes, which clearly reduced the average particle size.

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Based on these results, we can affirm that several sequential processes are generally involved in acid titration (Figure 7). Initially, there is a slight increase in

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turbidity owing to the formation of soluble “primary” complexes (pHc) between the biopolymers; upon further titration, the turbidity rapidly increases as soluble

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complexes transform into insoluble complexes (pHΦ1). The solution then achieves

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maximal turbidity at pHmax, where biopolymers achieve electric neutrality, which is followed by dissolution of the complexes at lower pH (pHΦ2) due to protonation of reactive groups on the biopolymer backbone (Liu et al., 2009). The insoluble

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complexes that form sediment can primarily be attributed to the degree of aggregation, although complexes become largely disassociated following protonation of the

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anionic groups on KC structures (pHΦ2), after which the biopolymers carry positive charges (de Kruif and Tuinier, 2001; Turgeon et al., 2003).

4. Conclusions

Turbidity analysis, particle size, ζ-potential measurements, and optical microscope were applied to investigate intermolecular interactions and phase behaviors in a MP-KC mixing system. The formation of both soluble and insoluble complexes between MP and KC were influenced by the pH of the system and the ratio 14

ACCEPTED MANUSCRIPT of these two biopolymers, resulting from electrostatic interactions and leading to different phase behaviors. Based on our results, we developed a phase diagram by plotting critical pH values and generated a schematic picture to highlight structure-forming transitions. The results presented here provide essential theoretical knowledge for control of MP-KC interactions, phase separation, and complex

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formation in food products. Acknowledgements

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Thanks for the financial support by the National Natural Science Funds (No.

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31571865 and No. 31371791).

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Alting, A. (2003). Cold Gelation of Globular Proteins. The Netherlands: Wageningen University. Braudo, E. E., Plashchina, I. G., & Schwenke, K. D. (2001). Plant protein interactions with polysaccharides and their influence on legume protein functionality A Review. Food/Nahrung, 45(6), 382-384 Çakir, E., & Foegeding, E. A. (2011). Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures. Food Hydrocolloids, 25, 1538-1546. Chung, C., Degner, B., & McClements, D. J. (2013). Controlled biopolymer phase separation in complex food matrices containing fat droplets, starch granules, and hydrocolloids. Food Research International, 54, 829-836. Da Silva, F. L. B., Lund, M., Jonsson, B., & Akesson, T. (2006). On the complexation of proteins and polyelectrolytes. The Journal of Physical Chemistry B, 110, 4459-4464. De Kruif, C. G., & Tuinier, R. (2001). Polysaccharide protein interactions. Food hydrocolloids, 15(4), 555-563. De Kruif, C. G., Weinbreck, F., & de Vries, R. (2004). Complex coacervation of proteins and anionic polysaccharides. Current Opinion in Colloid & Interface Science, 9(5), 340-349. DeFreitas, Z., Sebranek, J. G., Olson, D. G., & Carr, J. M. (1997). Carrageenan Effects on Salt-Soluble Meat Proteins in Model Systems. Journal of Food Science, 62(3), 539-543. Dickinson, E. (2008). Interfacial structure and stability of food emulsions as affected by protein–polysaccharide interactions. Soft Matter, 4(5), 932-942. Elmer, C., Karaca, A. C., Low, N. H., & Nickerson, M. T. (2011). Complex 15

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Zhang, T., Xu, X., Ji, L., Li, Z., Wang, Y., Xue, Y., & Xue, C. (2017). Phase behaviors involved in surimi gel system: Effects of phase separation on gelation of myofibrillar protein and kappa-carrageenan. Food Research International, 100, 361-368.

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Figure 1

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Fig. 1. Changes in the turbidity curve of MP-KC mixing system with ratio of 1:1 and total concentration of 0.05% (w/w), and homogenous MP and KC solutions were

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measured as controls.

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Figure 2

Fig. 2. Turbidity changes of MP-KC mixture influenced by biopolymer ratio R and pH with the total concentration of 0.05% (w/w). Square: R=1:1; Circle: R=5:1; Triangle towards up: R=10:1; Triangle towards down: R=15:1; Pentagram: R=20:1. 19

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Figure 3

Fig. 3. Phase behaviors and intermolecular interactions of MP-KC system effected by

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pH and R with total concentration of 0.05% (w/w) obtained by turbidimetric analysis

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from Figure 2. The solid line is the boundary of different states of phase behavior.

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Figure 4

Fig. 4. Particle sizes distributions for MP-KC system (R = 1: 1) effected by pH. The table inside was the mean values of the particle sizes of the mixing system at different pHs. MP and KC were also measured as controls. 20

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Figure 5

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Fig. 5. Zeta potential changes of MP, KC, and their mixtures with different biopolymer ratio during acid titration. Pentagram: MP; Square: KC; Triangle towards up: R1; Triangle towards down: R2; diamond: R3; Triangle towards left: R4; Triangle towards right: R5.

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Figure 6

Fig. 6. Light micrographs of MP-KC mixtures with the ratio of R=1:1 and total 21

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Figure 7

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Fig. 7. Schematic diagram of sequential processes involved in complex formation between MP-KC mixtures.

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Graphical abstract

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Highlights •Interactions and phase behaviors of myofibrillar protein and κ-carrageenan were studied. •A phase diagram was described against biopolymer mixing ratio and pH. •The complex formation was mainly driven by electrostatic attractive forces.

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