Journal Pre-proofs

Water-soluble myofibrillar protein–pectin complex for enhanced physical sta-

bility near the isoelectric point: Fabrication, rheology and thermal property

Xing Chen, Qingyang Qiu, Kaiwen Chen, Di Li, Li Liang

PII: S0141-8130(19)36195-1
DOI: https://doi.org/10.1016/j.ijbiomac.2019.10.003
Reference: BIOMAC 13383

To appear in: International Journal of Biological Macromole-

cules

Received Date: 6 August 2019

Revised Date: 18 September 2019
Accepted Date: 1 October 2019

Please cite this article as: X. Chen, Q. Qiu, K. Chen, D. Li, L. Liang, Water-soluble myofibrillar protein–pectin
complex for enhanced physical stability near the isoelectric point: Fabrication, rheology and thermal property,
International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.003

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Water-soluble myofibrillar protein–pectin complex for enhanced

physical stability near the isoelectric point: Fabrication,

rheology and thermal property

Xing Chen a, b, Qingyang Qiu a, b, Kaiwen Chen a, b, Di Li a, b, Li Liang a, b,

*,
a
State Key Laboratory of Food Science and Technology, Jiangnan

University, Wuxi, Jiangsu 214122, China

b
School of Food Science and Technology, Jiangnan University, Wuxi,

Jiangsu 214122, China

*Corresponding author, Telephone: +86(510)85197367.

E-mail address: xingchen@jiangnan.edu.cn (X. Chen);

vegetto0305@163.com (Q. Qiu); chenkaiwen1018@163.com (K. Chen);

495893059@qq.com (D. Li); liliang@jiangnan.edu.cn (L. Liang*);

Declarations of interest: none

ABSTRACT: This study tested the potential of forming soluble complex with

pectin (PEC) on enhancing physical stability of water-soluble myofibrillar protein

(WSMP) near the isoelectric point (pI, pH 5.00–5.50). After incorporation of PEC at

the mixing ratio of 10:1 and 5:1, WSMP suspension maintained transparent state at

0.05 wt% while a homogeneous monophase at 1.00 wt% around pI, indicating the

formation of soluble WSMP–PEC complex. When mixing the two biopolymers,

charge neutralization was observed, revealing the electrostatic attraction between

positively charged patches of WSMP and negatively charged carboxyl sites of PEC.

Steady shear results showed a reduced viscosity of WSMP–PEC complex when

dropping the pH to 5.00, this may be related to the declined biopolymer net charge

and water trapping. Oscillatory data suggest the formation of highly-interconnected

network in soluble WSMP–PEC complex, thus decreasing pH or biopolymer ratio can

enhance their interactions and thereby lead to stronger and more stable microstructure.

Thermal denaturation temperature of WSMP was significantly enhanced through the

formation of WSMP–PEC soluble complexes. Overall, complexation with PEC

improved the colloidal and thermal stability of WSMP around the pI, which

evidenced the potential of applying tailor designed protein-polysaccharide complex in

formulating muscle protein-based beverages.

KEYWORDS: Water-soluble myofibrillar protein; pectin; soluble complex.

1. Introduction

There is strong interest in utilizing proteins to facilitate the engineering for

fabrication of novel food products, such as meal replacement beverages or recovery

sports drinks [1-3]. The functionalities of food proteins largely determine the

effectiveness of their utilization in food production. Unequivocally, meat-derived

proteins represent an extraordinary nutritive ingredient in replenishing human needs

since they are distinguished by their abundance in essential amino acids and high

biological value [4, 5]. Compared with milk, egg white, and nut proteins [6], meat

proteins (e.g., myosin and actin) are non-allergic [7]. Besides, benefiting from their

high molecular weight (e.g., ~500 kDa for myosin), diverse side chain reactive groups

as well as the fibrous flexible structure, meat proteins are good candidates in the

design of tailor-made colloidal particles for delivering desirable viscoelasticity that

satisfy the various demands of food product manufacturers. Consequently, special

attentions have been drawn in applying muscle-based protein as ingredients into food

formula [8-10], probably due to the above-mentioned advantages along with high

quality protein, non-allergen and gluten-free claims. However, meat has not been

exploited as a supplementary protein ingredient to the same extent as milk or soybean,

mainly because of the poor functionality of myofibrillar protein (MP, approximately

50% of the total meat proteins) in water or at low ionic strength [11].

The most important functional properties related with protein fortified beverage

include its solubility (or colloidal stability), rheological behaviors and thermal

stability. In our previous study, high-pressure homogenization (HPH) was used to

selectively modify the structure of MP for improved solubility and thermal stability in

water without obvious loss of its rheological behaviors [8], which has opened up a

promising area for the R&D of muscle MP in semi-solid or liquid food system at low

ionic strength. However, the applications of MP in beverage formula are still

challenging. MPs are notoriously sensitive to environmental stress, such as the pH and

temperature. MP loses its net negative charge and has neutral charge, rendering the

weakest hydration around its isoelectric point (pI, around pH 5.50). When products

are acidified and heated, MP will quickly aggregate and be subjected to sedimentation

or gelation in the final products, which is absolutely a nuisance when a MP beverage

is formulated at pH 4–6 (an ideal pH range for beverage formula in terms of sensorial

defects) [1]. Therefore, novel strategies to enhance the colloidal stability of MP

aqueous solution are warranted.

The complexation of proteins with polysaccharides has been shown to be a

promising approach to improve protein colloidal stability at acidic environment

without affecting their structuring abilities [12]. An additional complexation with

hydrocolloids such as pectin can limit the aggregation of whey protein near the pI and

create soluble particles for beverage application [1]. Physical stability of pea protein

isolate (PPI) at acidic solution can be also enhanced upon the formation of soluble

complexes with pectin [2]. This complexation occurs from an electrostatic interaction

between anionic polysaccharides and positively charged surface patches of protein,

and it can be influenced by several physicochemical parameters in the system, such as

the type of biopolymer, biopolymer concentration, mixing ratio, pH, and charge
density. By judicious selection of these parameters, specific structures and functional

properties can be generated according to their usability in different food system [13].

Numerous studies have focused on the fabrication of protein–polysaccharide complex

for superior functional properties, such as milk proteins–pectin/sodium alginate [1,

14], egg proteins (lysozyme, ovalbumin)–-carrageenan/gum arabic [15, 16], plant

proteins (PPI, wheat gliadin)–pectin/gum arabic [2, 3]. However, the formation and

application of soluble complexes between meat-derived proteins (e.g., MP) and

polysaccharides have been scarcely explored yet. The main reason is due to the

presence of high salt content (> 2% or 0.5 M NaCl) usually used when solubilizing

and handling MPs (commonly known as salt–soluble proteins) during research or

processing. Under such a high ionic strength, the electrostatically driven formation of

complexes between proteins and polysaccharides can’t exist, or is too tiny to be

detected. In addition, MPs have slightly higher pI value (5.50) than that of WPI (5.20

for -lactoglobulin, 4.10 for -lactalbumin) [17] and PPI (4.05~4.60) [2, 18],

meaning that more basic side chains (lysines, arginines, and histidines) are

surface-exposed. Combined with their elongated flexible structure, MPs are supposed

to be more capable of forming complexes with potent charge polysaccharides via

electrostatic association or inter-molecular entangling, which however remains to be

determined.

It is assumed that the fabrication of protein–polysaccharide soluble complex might

be a good alternative to enhance the physical stability of water-soluble MP (WSMP)

at mildly acidic environment. In the present study, pectin (PEC), known as an linear
anionic polysaccharide containing predominately α-(1→4)-linked partially methyl

esterified D-galacturonic acid and rhamnogalacturonan units, was selected as the

model polysaccharide because of its tunable structure, widespread research and

application in acidic protein beverages [1, 2, 13]. The aim of this study was to

investigate the effects of WSMP/PEC mixing ratio (2:1–20:1), pH (7–3) and WSMP

concentration (0.05 and 1.00 wt %) on the formation of soluble complexes using

turbidimetric analysis, phase diagram, and -potential, and hereafter to determine the

rheological and thermal properties of selected biopolymer soluble complex. So far,

the literature on the complexation of muscle proteins with pectin is rather limited,

results gained from this research will provide a better understanding of MP soluble

complexes formation that can be applied in beverage industry.

2. Materials and methods

2.1. Materials

Chicken breast muscle (Pectoralis major) was collected at 36–48 h postmortem

from Sushi Food Co., Ltd. (Nanjing, Jiangsu, China). Muscle samples were vacuum

packaged, stored in a –20 °C freezer, and used within 4 days after slaughter. Citrus

pectin (product number A600685, MW 50–300 kDa, degree of esterification = 65 %,

residue < 0.3%, water < 12%) was purchased from Sango Biotech Co. (Shanghai,

China). Other chemicals were at least of analytical grade and purchased from Sigma–

Aldrich (St. Louis, MO, USA).

2.2. Preparation of WSMP and PEC stock solution

WSMPs were prepared by a procedure described previously [19-21]. In brief, the

mince of chicken breast muscle was homogenized and washed with series of sodium

phosphate buffer (pH 7.00) containing 25~100 mM NaCl and 5 mM EDTA, followed

by triplicate Milli-Q water washing. The resultant protein pellets were re-suspended in

Milli-Q water at the appropriate concentration and were subsequently subjected to

HPH treatment at 69 MPa for two cycles by the AH-2010 Nano Homogenize Machine

(ATS Engineering Inc., ON, Canada). The interaction cell and the outlet were covered

with cold water circulating system to maintain the outlet temperature below 20 °C.

The HPH-treated samples were then cold centrifuged at 8000 g for 10 min. The

obtained supernatant hereafter was termed as WSMP [20]. WSMPs were adjusted to

pH 7.00 and then stored at refrigerator overnight prior to use.

PEC stock solution was prepared by dispersing pectin in Milli-Q water under

stirring (500 rpm) for 12 hours at room temperature to promote hydration. The stock

solution was then adjusted to pH 7.00 and stored at 4 ℃ prior to use.

2.3. WSMP–PEC mixture

To determine the formation of WSMP–PEC soluble complexes as a function of pH,

biopolymer mixing ratio and WSMP concentration, two groups of WSMP–PEC

mixture were prepared: (i) low WSMP group with a fixed concentration of 0.05 wt%

but variable final PEC concentrations from 0.0025 to 0.025 wt%; (ii) high WSMP

group (a fixed concentration of 1.00 wt%) mixed with PEC varying from 0.05 to 0.50

wt%. Biopolymer solutions were prepared by adding 15 mL of pectin stock solutions

into the 35 mL of WSMP stock solution to obtain the following WSMP:PEC mixing

ratios: 20:1, 10:1, 5:1 and 2:1. For the turbidimetric titration, the WSMP–PEC
mixtures with low WSMP were prepared by adjusting the pH from 8.00 to 2.00 under

magnetic stirring [2]. The phase behavior of WSMP–PEC mixture having high

WSMP (1.00 wt%) was investigated by visual observation of mixtures using a digital

camera after adjusting the pH from 7.00 to 3.00. Solution pH was lowered using a

gradient of HCl concentrations to minimize any dilution effect according to the

procedure described by Klemmer, Waldner, Stone, Low and Nickerson [18].

2.4. Turbidimetric analysis

Turbidimetric acid pH titrations were performed to identify the critical pH

transition point (pHsol) for the formation of soluble complex using the method of Lan,

Chen and Rao [2]. Turbidity of samples (low WSMP group) was measured in 3.5 mL

disposable cuvettes (1 cm path length) at 600 nm using a UV-visible

spectrophotometer (UV-1800, Shimadzu Co., LTD., Kyoto, Japan). Individual WSMP

and PEC solutions were used as controls at 0.05 wt% and 0.025 wt% concentrations,

respectively. All turbidity profiles were prepared in triplicate, and associated critical

pH values were reported as the mean value ± standard deviation.

2.5. -potential measurement

To determine the overall surface charge, -potential measurements of WSMP, PEC,

and their mixtures were performed at 25 °C using a NanoBrooker Omini Particle Size

Analyzer (Brookhaven Instrument, New York, USA) fitted with a BI-SCP sample cell.

Exactly 0.05 wt% WSMP and 0.025 wt% PEC solutions were also prepared as

controls.

2.6. Rheological measurement

 Rheological tests of selected soluble WSMP–PEC complexes at various ratios (10:1

and 5:1) and pH values (6.00, 5.50 and 5.00) fitted with a parallel plate geometry (40

mm diameter) were measured at 25 °C using a HAAKE MARSTM Rheometer

(Thermo Scientific, MA, USA). In general, samples (~2 mL) were loaded in the fixed

gap (0.5 mm) between the rheometer bottom plate and the parallel plate geometry,

which were subsequently subjected to steady shear, strain sweep and frequency sweep

test, respectively. A thin layer of low-viscosity silicone oil was placed on the

peripheral surface of the solution held between the plates to prevent moisture loss

from the samples during measurement. Individual WSMP and PEC samples were

used as controls.

2.6.1. Apparent viscosity measurement

The apparent shear viscosity () was measured based on the method described

previously [20]. After an equilibration time of 30 s, the apparent viscosity was tested

within a linearly increased shear rate range of 0.1–100 s−1. The apparent viscosity of

the samples versus logarithmic shear rate was recorded.

2.6.2. Strain sweep

Strain sweep was commonly the initial step of small amplitude oscillatory shear

measurements (SAOS), aiming at determination of the linear viscoelastic region

(LVE). Herein, the elastic modulus (G′) and viscous modulus (G″) over a range of

0.1–100% strain sweep () in controlled rate mode at 1 Hz were recorded. The critical

strain (c, limit of linearity) was determined accordingly [22] and expressed as the

mean of triplicate treatments.

2.6.3. Frequency sweep

Frequency sweep was carried out over a range of 0.1–100 Hz frequency () at 1%

strain to investigate the viscoelastic behaviour of soluble complexes. The degrees of

frequency dependence of G′ (1) and G″ (2) were fitted by the power law constitutive

equation as follows:

G′ = k′𝜔 𝑝

(1)

G′′ = k′′𝜔𝑞

(2)

where k′ and k′′ (Pas) are intercepts; p and q are the viscoelastic components of

elastic and viscous moduli and  is oscillatory frequency (Hz) [22]. The values of p

and q were reported as the averages of triplicate treatments.

2.7. Differential scanning calorimetry (DSC)

Thermal stability of WSMP and its selected soluble complexes was analyzed using

a Q2000 differential scanning calorimeter (TA Instruments Ltd., DE, USA) as

described previously [23]. Accurate weighted samples (10 mg/mL protein, 16–18 mg)

hermetically sealed in aluminum pans were subjected to 20–120 °C thermal scan at a

5 °C/min rate. An empty aluminum pan was used as reference. The temperature

maximum (Tmax) for transition was estimated using Universal analysis software

(Version 1.2N) obtained from the TA Instruments.

2.8. Statistical analysis

Three independent trials were carried out for each treatment. The analyses of data
were performed using one-way ANOVA and Duncan’s multiple range tests when

necessary with the Statistical Analysis System (SAS Institute Inc., Cary, NC, USA).

A P < 0.05 significance level was used to determine the differences between the

treatments.

3. Results and discussion

3.1. WSMP–PEC soluble complexes formation

3.1.1. WSMP–PEC soluble complexes formation at low WSMP concentration

Monitoring the changes in optical density (OD) during a pH acid titration is a

practical tool in investigating the complex formation of protein–polysaccharide

biopolymer mixtures at diluted concentrations. As shown in Fig. 1A, individual PEC

solution remained transparent and showed no remarkable changes across the entire pH

range, suggesting that pectin did not form aggregates to scatter light during the pH

titration process. Since PEC is an anionic biopolymer containing carboxyl functional

groups (pKa=3.5), a relatively strong electrostatic repulsion between molecules would

be reasonable to be existent, which can prevent them from forming aggregates. The

OD of individual WSMP solution remained relatively low under both low pH (~3.30)

and high pH (6.40–7.00) conditions. However, a rapid rise in OD was observed as pH

approached 4.00–6.00, which is thought to be attributed to an increase in protein–

protein aggregation associated with reduced charge repulsion between neighbouring

protein molecules near MP’s pI (-potential = 0 mV, Fig. 1C). The addition of PEC to

a WSMP solution caused a leftwards skewed turbidity profiles, indicating that there

was an intermolecular interaction between WSMP and PEC [2]. For instance, WSMP
had a starting pH point of 6.40 for turbidity elevation whereas, for WSMP/PEC (10:1)

mixture, the rise in turbidity began at a reduced pH value (around 5.00, Fig. 1A).

Mostly, complex formation between anionic polysaccharides and positively-charged

patches of proteins is expected to initiate at the conditions between neutral pH and the

pI of protein [24], lowering pH strengthens the electrostatic interaction as a result of

more positive charges of the protein. From this point of view, we speculated an

incipient binding between WSMP and PEC, therefore soluble and stable complexes

were formed in WSMP/PEC (10:1) mixture at pH 6.40–5.00 (Fig. 1A). With further

decreasing pH, the formation of insoluble complexes occurred, the turbidity of

WSMP–PEC mixtures (10:1) increased dramatically and the turbidity peaked at pH

4.20 (Fig.1A). This rapid increase in turbidity could be ascribed to two reasons: (i) As

pH decreased, stronger interactions between two polyelectrolytes driven by both

electrostatic attraction and hydrogen bond took place [25], this can ultimately lead to

complete charge neutralization as well as fewer negative charges carried on WSMP–

PEC complexes, thereby the electrostatic repulsive forces were expected to decrease,

and the complexes maybe ease to approach and associate to a greater extent. (ii)

Entanglement of polysaccharide chains and fibrous structure of MP molecules may

also contribute to the coacervation process, resulting in enhanced turbidity [26].

Overall, the shift to reduced pH with the addition of PEC suggests structures with

increased stability near pI relative to WSMP in the absence of PEC.

As the WSMP/PEC mixing ratio decreased from 20:1 to 2:1 (fixed concentration of

protein, PEC levels increased), the pH-dependent turbidity profiles as well as the
delay of aggregate growth shifted to more acidic pH (Fig. 1A). It seemed that higher

levels of PEC favored greater formation of soluble WSMP–PEC complexes to

withstand more acidic pH environment. Similar trends of shifted profiles were also

reported in the bovine serum albumin–PEC [27], PPI–PEC [2] and ovotransferrin–

PEC complexes [24]. To indicate the formation of WSMP–PEC soluble complexes,

the critical pH transition point (pHsol) from soluble complex to insoluble complex was

selected based on the above results. It is defined as the pH value under which the

inflexion point in the optical density versus pH curve is occurred (Fig. 1A). With

mixing ratio decreased, an obvious shift in the pHsol of WSMP–PEC complex from

pH 6.21 at 20:1 biopolymer ratio to pH 4.45 at 2:1 was observed (Fig. 1B). In other

words, higher PEC concentration increased the stability of WSMP at lower acidic pHs.

This phenomenon can be explained by assuming that intermolecular complexes

formed between WSMP and PEC will provide more negative charges on protein

surface (as evidenced in Fig.1C) to prevent protein aggregation at acidic pH [2]. It

was noteworthy that the presence of PEC at a concentration no less than 0.1%

(WSMP/PEC mixing ratios of 10:1 or lower) was necessary to reduce the aggregation

of WSMP around the pI (pH 5.00–5.50), presumably due to its ability to form a

soluble complex bearing sufficient negative charge in hindering intermolecular

association.

The electrical charge on the biopolymer suspensions was also investigated. As

displayed in Fig. 1C, with pH decreasing from 7.00 to 3.00, -potential of individual

WSMP elevated from −28.7 mV to 37.6 mV with the point of zero being around pH
5.30 (pI), the transformation from negative charge to positive change on WSMP

solution is mainly due to the protonation of carboxyl groups and protein amine during

the acid titration [14]. Because of negative carboxyl groups distributed on the

backbone of PEC, the individual PEC carried a negative charge throughout the entire

studied pH range and its -potential value increased from -45.2 mV to -6.0 mV (Fig.

1C) as a result of declined ionization of carboxyl groups during acidification. The net

-potential in the mixed WSMP/PEC system was intermediate between that of the

individual WSMP and PEC values within the pH range tested, indicating that the

charge neutralization occurred through electrostatic binding between anionic carboxyl

groups on the PEC and cationic amino groups on the protein surface. As the ratio of

WSMP to PEC decreased (20:1 to 2:1), the -potential of WSMP/PEC mixture

became more negative (Fig. 1C), this result is expected because, by decreasing the pH,

protein side chains would have more cationic amino groups (e.g., −NH3+), thus more

PEC molecules can bind to the protein surface [28], producing greater negative

charges. It was noted that the pH points where complete charge neutralization

occurred (-potential=0 in Fig. 1C) were consistent to the corresponding pH values

responsible for maximum turbidity (Fig. 1A). This finding is in agreement with

previous studies on ovotransferrin–pectin and milk proteins–sodium alginate

complexes, they reported that strongest protein−polysaccharide interactions shown in

spectrophotometry appeared when the electrical charges of the mixtures were nearly

neutralized [14, 24]. Therefore, it is inferred that the formation of insoluble and

soluble protein–polysacharide complexes was in large part caused by the electrostatic

attractions between the two biopolymers [14]. The -potential supported the result of

OD measurements on the soluble complexes, which suggested that the complexation

of highly negative-charged PEC could cause sufficient electrostatic repulsion to delay

protein aggregation, thereby improving colloidal stability of WSMP–PEC suspension

at pH 6.00–5.00. It was noteworthy to highlight that the side branches of adsorbed

PECs may also hinder approaching of proteins particles by steric repulsion [25].

3.1.2. WSMP–PEC soluble complexes formation at high WSMP concentration

Identification of soluble complex using turbidimetric analysis is fairly easy to

achieve in diluted biopolymer systems, however it is not applicable for high

concentration of binary protein/polysaccharide system because of the high initial OD

of biopolymer mixture, thus the turbidimetric findings may not accurately match the

real food system in which proteins and polysaccharides are usually present with

relatively high content. Herein, the segregative and associative phase behaviors of

WSMP/PEC mixtures at high protein content (1.00 wt%) were directly visualized as

the function of pHs and mixing ratios in Fig. 2. For the individual WSMP (1.00 wt%),

phase separation occurred during the pH range of 6.00–4.00 owing to the aggregation

and precipitation of proteins around the pI, which was consistent with the results of

OD measurements (Fig. 1A). After mixing with PEC, the pH value associated with

phase separation region moved toward acidic condition, and one phase region

expanded as mixing ratio decreased from 20:1 to 10:1 (Fig. 2). Analogous phase

behaviors as function of biopolymer ratio were observed in PPI–PEC [2] and milk

proteins– tragacanth/persian gum mixtures [25]. Phase segregation in the present

biopolymer suspensions is supposed to be caused by the associations of proteins as

well as WSMP–PEC coacervation [25]. These results suggested that the presence of

PEC can inhibit biopolymers aggregation and stabilize their solution at low pH

conditions. It has been reported that electrostatic binding of anionic carboxyl groups

on PEC to cationic amino groups on protein surface gave rise to the formation of

soluble complexes which had sufficient negative charge to repel each other thus

preventing the aggregation [2, 24, 29]. Further reducing the biopolymer ratio to 5:1 or

lower (PEC content increased) promoted the phase separation at pH 3.50 and 3.00

(Fig. 2), probably as a consequence of intensified WSMP–PEC coacervation. Notably,

in the case of WSMP/PEC suspension at 2:1 mixing ratio, phase separation occurred

around neutral pH with a white precipitate layer and an opaque upper layer (Fig. 2).

This phase separation at high concentration of biopolymers was distinctive to the

results of turbidity that observed in diluted systems (Fig. 1A), and it could be

attributed to thermodynamic incompatibility effect between the WSMP and PEC [30].

Based on the above results, at the mixing ratios of 10:1 and 5:1, WSMP–PEC had a

low turbidity at diluted condition as well as homogenous monophase behavior at high

biopolymer content under pH 6.00–5.00, indicating the formation of stable complex

around pI of WSMP. Hence, soluble WSMP–PEC complexes with the ratios of 10:1

and 5:1 at pH 6.00, 5.50 and 5.00 were selected for the further research.

3.2. Rheological properties of WSMP–PEC soluble complexes

3.2.1. Flow behavior

As can be seen in Fig. 3, the apparent viscosity of all tested samples presented a
constant decrease with increasing shear rate, indicating a shear thinning behavior. The

shear thinning behavior of WSMP has already be early recognized in literature [31,

32]. At a low rate of shearing, rod-like myosin (the major component of MP) or

swelling myofibrils fragments in WSMP tend to overlap and entangle, causing a high

viscosity through the steric hindrance and internal friction within the protein chains.

When increasing the shear rate, the connections between protein chains can be

disrupted, and the asymmetrically dispersed molecules would more likely align

themselves with the shear planes, thus weakening the frictional resistance [8].

The soluble WSMP–PEC complex demonstrated higher apparent viscosity than that

of WSMP (1.00 wt%) or PEC (0.20 wt%) alone, which is in accordance with previous

studies on WPI–PEC [1], canola protein–PEC [33] and PPI–PEC [2]. This can be

interpreted by the attachment of highly charged and branched PEC on protein surfaces,

which could provide strong repulsion and steric hindrance between particles,

increasing the resistance against flow. Meanwhile, the presence of hydrogen bonding

between the neighboring molecules upon the formation of soluble complexes might

also contribute to the enhanced viscosity [25]. As a result, the viscosity can be further

increased by complexation with greater PEC via reducing the WSMP/PEC mixing

ratio from 10:1 to 5:1 (Fig. 3A).

Apparent viscosity of soluble complexes as affected by the pH was also studied. As

shown in Fig.3B, dropping the pH from 6.00 to 5.00 led to continuous decline in

viscosity of soluble complexes. This was contrast to the result of Lan, Chen and Rao

[2] who observed that apparent viscosity of soluble PPI–PEC complex at pH 5.00 was
higher than that at pH 6.00. Although higher amount of branched PEC can be

absorbed on the protein surface by decreasing the pH to induce greater formation of

soluble complex, the reduced net charge of WSMP–PEC particle (as observed in Fig.

1C) might have largely weakened the electrostatic repulsion between complexes, thus

resulting in a more flexible structure responsible for the lower viscosity. Moreover,

being a fibrous structure with large molecular weight (higher than that of PEC

molecule), the viscosity of MP was reported be closely related to swelling state of

myofibrils in WSMP: Moving the pH apart from pI caused strong electrostatic

intermolecular repulsion, which make hydrophilic groups available to entrap water,

myofibrils can thereby become swelling state, increasing the intrinsic friction of the

system [34]. In the present system, diminishing the pH might not be conductive to

water retention and biopolymer swelling, hence the viscosity of soluble WSMP–PEC

complex was reduced.

3.2.2. Strain sweep

Typical strain sweep curves of soluble WSMP–PEC complex was given in Fig. 4.

For all samples, the G′ dominate over the G′′ in the LVE region, indicating the elastic

portions was the primary response to the oscillation. The G′ and G′′ values of WSMP–

PEC complex were regularly higher than the counterparts of individual WSMP and

PEC suspensions during the entire strain sweep, suggesting a stronger structural

strength of biopolymers presented in WSMP–PEC complex. The appreciable increase

in G′/G′′ has been reported to be attributed to the protein/polysaccharide interactions

[22]. This is supported by the observation that the complexes formed in the presence
of higher PEC concentration (at mixing ratio of 5:1) exhibited a greater G′/ G′′

compared with those formed with a low PEC content (10:1) (Fig. 4A). The step

reduction in pH from 6.00 to 5.50 could improve the moduli of WSMP–PEC

progressively (Fig. 4B). Indeed, as the pH decreased, the positively charged patches

of WSMP gradually increased, extensive intermolecular interactions via both

electrostatic attraction as well as the hydrogen bonding between biopolymers can take

place. Together with intermolecular entanglement, it was assumed that WSMP might

be more liable to interact with PEC at junction zones and form a stronger viscoelastic

network, producing higher response to the oscillation. This was also in accordance

with the result of critical stress (Table 1). The critical stress (c) is a stress in which G′

decreases sharply depending on the molecular architecture of the food polymer

molecules [35]. It is associated with the network stability and gel deformability. It

was found the WSMP–PEC complex was much more strain resistant (c=7.74% for

10:1, 15.97% for 5:1) than WSMP (c=6.16%) and PEC (c=2.33%) alone (Table 1),

revealing more stable network structures ascribed probably to the establishment of

associated WSMP/PEC matrix. When decreasing the pH towards the pI of protein, c

went up from 3.41% to 15.81% (Table 1). It seemed that greater complexation of PEC

with WSMP could stabilize biopolymer microstructure, which significantly reduced

the strain sensitivity.

3.2.3. Frequency sweep

Frequency sweep provides suitable knowledge about network structure. As shown

in Fig. 5, all samples established an interconnected gel-like network structure in

which G′ displayed a modulus higher than G′′ over the entire frequency range, and the

two curves were almost parallel to each other. The WSMP–PEC displayed higher

moduli than that of individual WSMP and PEC, and the values can go up by

decreasing the mixing ratio (Fig. 5). It seemed that the increase in PEC (mixing ratio

decreased) favored the formation of strong gel-like microstructure. Similar results in

protein–polysaccharide complexes have been reported including bovine serum

albumin (BSA)–pectin [27] and -Lactoglobulin–pectin [36]. They declared that the

interactions between protein molecules and PEC chains were responsible for the

enhanced viscoelastic behavior [16, 22]. This is also supported by the report of

Espinosa-Andrews, Sandoval-Castilla, Vázquez-Torres, Vernon-Carter and

Lobato-Calleros [37] who found that strong electrostatic interactions in complex

coacervates was positively related to a dense network structure observed by scanning

electron microscopy, a more dense network led to higher viscoelastic moduli. At

lower WSMP/PEC mixing ratio (5:1 v.s. 10:1), higher amount of actual pectin would

take part in complexation, resulting in a stronger intermolecular WSMP/PEC network.

In this regard, it was expected that a reduction of pH caused an obviously shift of

moduli towards higher values and induced a more entangled network (Fig. 5b).

Because decreasing pH can cause changes to the electrostatic charge of biopolymer

molecular surfaces and increase positive charge on the WSMP residue, which led to

higher tendency of forming WSMP–PEC complexes as discussed above. Similar

change on G′/G′′ as function of pH was observed by Ghorbani Gorji, Ghorbani Gorji,

Mohammadifar and Zargaraan [38] who discovered that a tighter complexation

structure between sodium caseinate and gum tragacanth was favored by decreasing

pH down to ∼4.04. It should be noted that the self-aggregation of WSMP may not be

a dominant contributor to their strengthened network as PEC has been shown to

largely suppress WSMP aggregations when dropping the pH down to pI in WSMP–

PEC systems (Fig. 1A and Fig. 2).

The power law has been used to model the moduli–frequency data of samples.

According to R2 values, all samples showed a good fitting to the model (Table 2). The

p and q values, which represent the slope in a log–log plot of G′ and G′′ versus

frequency (), respectively, are commonly used to indicate the strength and the gel

structure formed. In general, shifting p values towards 0 indicates the transformation

of biopolymer suspensions from weak physical network to strong covalent gel [39].

WSMP–PEC solution exhibited significantly higher value of p and q than those of

individual WSMP and PEC (Table 2), indicating that viscoelastic property of WSMP–

PEC were less dependent on frequency than that of WSMP and PEC alone. And there

were more initial biopolymer intermolecular interactions or entanglements in WSMP–

PEC solutions, these interactions increased with decreasing WSMP/PEC mixing ratio

(Table 2). The p value of WSMP–PEC was almost high at pH 6.00 and it decreased

from 0.4436 to 0.2150 by reducing pH level to 5.00 (Table 2), this indicated the more

liquid-like response at pH value of 6.00 and strong WSMP/PEC complexation

structures at pH 5.00. The intermolecular interactions between WSMP and PEC were

probably greater at lower pH and consequently fabricated a stable network structure

that was less dependent to the oscillation frequency.

3.3. Thermal property

The thermal behaviors of WSMP–PEC formed at different pHs were presented in

Fig. 6 and Table 3. The DSC thermograms of WSMP at pH 6.00 showed three major

endothermic peaks at 56.58 C (Tmax1), 71.92 C (Tmax2) and 80.08 C (Tmax3),

respectively (Fig. 6A and Table 3). These peaks were reported to correspond to the

transitions of heavy meromyosin (myosin head), light meromyosin (myosin tail) and

actin, respectively [40]. The three transition temperatures shifted towards higher

values after complexation of PEC at both pH 6.00 and 5.50, revealing an enhanced

thermal stability of WSMP subunits. It is reported that complexation between protein

and polysaccharide (e.g., soluble complexes and complex coacervations) increases

protein stability against thermal denaturation [2, 41]. The increase in thermal stability

of myosin and actin was presumably the consequence of conformational preservation

of PEC molecules covered on corresponding protein domains. At pH 5.00, the

formation of WSMP–PEC complex was thought to be more extensive, which resulted

in the disappearance of the first peak, increment of Tmax2 from 75.08 to 76.92 and

recovering of the third transition peak (Fig. 6C, Table 3). It seemed that structural

rearrangement occurred on myosin head and actin by complexation with PECs at this

condition, leading to a new network though the changes have not yet been designated.

4. Conclusions

The potential of forming soluble complex with PEC for enhanced physical stability

of WSMP around the pI was tested in the present study. At a diluted WSMP

concentration, WSMP–PEC soluble complexes near the pI (pH 5.00~5.50) can be

formed at biopolymer ratios of 10:1 or less. As WSMP–PEC mixing ratio decreased

from 20:1 to 5:1, the pH of soluble complexes formation moved towards acidic values.

However, at a high WSMP concentration (1.00 wt%), soluble complexes around the

pI only existed at biopolymer mixing ratios of 10:1 and 5:1. -Potential results

showed a charge neutralization between biopolymers, indicating the electrostatic

attractive interactions between the cationic patches (e.g., -NH3+) of WSMP and the

carboxyl groups of PEC. As WSMP/PEC mixing ratios decreased, -potential of

WSMP–PEC mixture became more negative due to the greater negatively charged

PECs attached on WSMP. Rheological results of soluble WSMP–PEC complexes

suggested that all samples displayed a shear thinning flow behavior. The viscosity of

soluble complexes formed at pH 5.50 elevated with reducing the mixing ratio from

10:1 to 5:1 whereas the one formed with a fixed ratio of 5:1 declined as pH reduced

from 6.00 to 5.50. The lowered surface charge as well as the loss of water trapping

ability as a result of pH dropping contributed to the reduced viscosity of soluble

WSMP–PEC complex. Soluble WSMP–PEC complex showed a

highly-interconnected network structure, which mainly resulted from the

intermolecular entangling and electrostatic interactions between WSMP and PEC

molecules. The decrease in pH and mixing ratio led to the formation of more stable

and compact microstructure of biopolymer mixtures. Myosin and actin in WSMP

were shown to be more resistant against thermal denaturation after complexation with

PEC. Overall, the present work confirmed the fabrication of soluble WSMP–PEC

complex as a promising way in improving the physical stability of WSMP at mildly

acidic environment, and provided the knowledge to suggest the implementation of

tailor designed protein–polysaccharide complex for developing muscle protein-based

beverages with desirable properties.

■ Acknowledgments

This work was financially supported by the National Science Foundation of China

[grant numbers 31901611], the Fundamental Research Funds for the Central

Universities [grant numbers JUSRP11970] and the Self-Initiated Research Project of

State Key Laboratory of Food Science and Technology, Jiangnan University [grant

numbers SKLF-ZZA-201906].

■ ORCID

Xing Chen: 0000-0002-8730-8029

Li Liang: 0000-0001-9584-6778

■ Notes

The authors declare no competing financial interest.

■ Author contributions

Xing Chen drew all tables and figures, and drafted the manuscript. Qingyang Qiu and

Kaiwen Chen contributed to the analysis and interpretation while Di Li and Li Liang

contributed to the discussion of the content, preparation of the manuscript and

checking of the language.

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■ Figure captions

Fig. 1 (A) Mean turbidity profiles of individual WSMP (0.05 wt%), PEC (0.025 wt%)

and WSMP/PEC mixed suspensions as functions of pH and mixing ratio. (B) Critical

pH transition points (pHsol) as a function of biopolymer mixing ratio obtained from

turbidity results. (C) -Potentials of individual WSMP (0.05 wt%), PEC (0.025 wt%)

and WSMP/PEC mixed biopolymer suspensions as functions of pH and mixing ratio.

Fig. 2 The segregative and associative phase behaviors of WSMP/PEC mixtures in

the presence of 1.00 wt% WSMP as affected by pH and mixing ratio.

Fig. 3 Apparent viscosity () versus applied shear rate of soluble WSMP-PEC

complexes as the function of (A) mixing ratios formed at pH 5.50 or (B) pHs at

mixing ratio of 10:1. Individual WSMP (1.00 wt%) and PEC (0.20 wt%) were used as

controls.

Fig. 4 Storage modulus (G) and loss modulus (G) versus strain (%) for soluble

WSMP-PEC complexes as the function of (A) mixing ratios formed at pH 5.50 or (B)

pHs at mixing ratio of 10:1. Individual WSMP (1.00 wt%) and PEC (0.20 wt%) were

used as controls.
Fig. 5 Storage modulus (G′) and loss modulus (G′′) versus angular of frequency at

strain = 1% for soluble WSMP-PEC complexes as the function of (A) mixing ratios

formed at pH 5.50 or (B) pHs at a mixing ratio of 10:1. Individual WSMP (1.00 wt%)

and PEC (0.20 wt%) were used as controls.

Fig. 6 DSC thermogram of individual WSMP and soluble WSMP-PEC complex

formed at (A) pH 6.00, (B) pH 5.50 and (C) pH 5.00 with a mixing ratio of 10:1.
■ Figure graphics
(A

(B

(C

Fig. 1 Xing Chen et al.

 p
7. 6. 5. 5. 4. 4. 3. 3.
H
0 0 5 0 5 0 5 0

WS
MP

WSMP/P
EC
(20:1)

WSMP/
PEC
(10:1)

WSMP/
PEC
(5:1)
WSMP/
PEC
(2:1)

Fig. 2 Xing Chen et al.

(A

(B

Fig. 3 Xing Chen et al.

(A

(B

Fig. 4 Xing Chen et al.

(A

(B

Fig. 5 Xing Chen et al.

(A)

(B)

(C)

Fig. 6 Xing Chen et al.

■ Tables

Table 1 Effects of biopolymer mixing ratio and pH on critical stress (c) of soluble
WSMP-PEC complex estimated from the strain sweep data*.

Mixing ratio ** c (%) pH *** c (%)

6.16 ± 0.17 c 3.42 ± 0.91 c
1.00 %WSMP WSMP-PEC (6.00)
0.20 % PEC 2.33 ± 0.10 d WSMP-PEC (5.50) 7.76 ± 0.12 b
WSMP-PEC (10:1) 7.74 ± 0.21 b WSMP-PEC (5.00) 15.81 ± 0.82 a
WSMP-PEC (5:1) 15.97 ± 0.17 a
* Means (n = 3) within the same column sharing no common letters (a−d) differ significantly (P < 0.05).

** Individual WSMP (1.00 wt%) and PEC (0.20 wt%) suspensions were used as controls. A fixed pH (5.50) was adopted to form

the soluble complex.

*** A mixing ratio of 10:1 was used to form the soluble complex.
 Table 2 Effects of biopolymer mixing ratio and pH on the frequency dependencies of
G′(p) and G′′(q) for soluble WSMP-PEC complex estimated from the frequency sweep
data*.

Mixing
p R2 q R2 pH*** p R2 q R2
ratio**
1.00 %WSM 0.4947±0.0 0.96 0.4640±0.0 0.99 0.4436±0.0 0.992 0.4446±0.0 0.9
6.00
P 073b 36 073b 66 237a 6 079a 955
0.5516±0.0 0.99 0.5547±0.0 0.99 0.3847±0.0 0.994 0.3837±0.0 0.9
0.20% PEC 5.50
180a 69 238a 71 123b 4 138b 942
WSMP-PEC 0.3882±0.0 0.99 0.3834±0.0 0.99 0.2150±0.0 0.992 0.2067±0.0 0.9
5.00
(10:1) 091c 44 079c 42 089c 6 055c 961
WSMP-PEC 0.2157±0.0 0.99 0.2064±0.0 0.99
(5:1) 059d 26 074d 61

* p and q values were obtained by fitting the G′ and G′′ in power law model (1) and (2), respectively. Means (n = 3) within the same

column sharing no common letters (a−d) differ significantly (P < 0.05).

** Individual WSMP (1.00 wt%) and PEC (0.20 wt%) suspensions were used as controls. A fixed pH (5.50) was adopted to form the

soluble complex.

*** A mixing ratio of 10:1 was used to form the soluble complex.

Table 3 Effects of forming soluble complex with PEC at different pHs on the thermal
properties of WSMP*.

pH 6.00 pH 5.50 pH 5.00

Tmax1 Tmax2 Tmax3 Tmax1 Tmax2 Tmax3 Tmax1 Tmax2 Tmax3
(C) (C) (C) (C) (C) (C) (C) (C) (C)
WSMP 57.92±0 68.58±0 78.17±0 56.58±1 71.92±1 80.08±1 54.83± 75.08±0
NP
.21b .55b .62b .21b .13b .22b 0.64 .63b
WSMP- 58.92±0 70.54±0 80.17±0 60.83±0 73.67±0 84.58±1 76.92±0 84.167±
NP
PEC .12a .42a .57a .64a .54a .24a .24a 0.65

* The maximum transition temperatures (Tmax) of endothermic peak were obtained from the DSC data. The Means (n = 3) within the same

column sharing no common letters (a−b) differ significantly (P < 0.05). NP: No peak was obtained. Soluble WSMP-PEC complexes were

formed at a mixing ratio of 10:1.

1
■ Highlights

 Water-soluble myofibrillar protein–pectin complex was formed.

 Enhanced physical stability around protein pI can be obtained at 1 wt% protein.

 Viscosity increased with pectin addition, but decreased with pH dropping.

 The elastic network may be due to the electrostatic complexation and entangling.

 Denaturation temperature of myosin and actin can be increased by forming complex.