Microstructure and rheology design in protein–protein–polysaccharide composites

Accepted Manuscript Microstructure and rheology design in protein–protein–polysaccharide composites Hassan Firoozmand, Dérick Rousseau PII:

S0268-005X(15)00153-8

DOI:

10.1016/j.foodhyd.2015.04.003

Reference:

FOOHYD 2946

To appear in:

Food Hydrocolloids

Received Date: 15 September 2014 Revised Date:

4 March 2015

Accepted Date: 5 April 2015

Please cite this article as: Firoozmand, H., Rousseau, D., Microstructure and rheology design in protein– protein–polysaccharide composites, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

Graphical Abstract

AC C

EP

Microstructure of gel consisting of 6 wt % gelatin mixed with 6 wt % starch and 2 wt% wheat protein isolate

ACCEPTED MANUSCRIPT

1 2

Microstructure and rheology design in protein– protein–polysaccharide composites

RI PT

3 4 5 6

8

SC

7

Hassan Firoozmand and Dérick Rousseau*

M AN U

9 10 11 12

14 15 16

TE D

13

Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, ON, M5B 2K3, CANADA

*Corresponding author: Tel: +1-416-979-5000 ext 2155; Fax: +1-416-979-5044

18

E-mail address: [email protected]

AC C

19

EP

17

1

ACCEPTED MANUSCRIPT

Abstract

2

The microstructure and rheology of protein–protein–polysaccharide composites were explored, with

3

wheat or soy protein used to modulate the microstructure and rheological properties of both gelatin and

4

gelatin + starch gels. The extent of phase separation typically associated with protein-polysaccharide

5

mixtures was greatly curtailed in the presence of either plant-based protein. The negatively-charged soy

6

protein generally increased the elastic modulus of the gelatin + starch gels whereas the positively-charged

7

wheat protein decreased it. Unique rheological behaviour was observed when both starch and 6 wt%

8

plant-based protein were present, as the phase-separated gels became soft and flowable.

9

Microstructurally, addition of soy protein to gelatin led to the presence of coacervates whereas the wheat

10

protein aggregated within the continuous gelatin phase. In gelatin + soy + starch mixtures, the gelatin and

11

soy remained mixed as the continuous phase and starch existed as the discontinuous phase. In gelatin +

12

wheat + starch mixtures, a complex multiple phase-separated microstructure was evident as the three

13

biopolymers appeared distinct. Overall, these results demonstrated that plant-based proteins may be used

14

as structure modifiers to generate novel phase-separated gels with diverse rheological properties.

15

Keywords: Phase separation; Rheology; Gelatin; Starch; Wheat protein isolate; Soy protein isolate;

16

Thermodynamic incompatibility; Multicomponent gels.

M AN U

SC

RI PT

1

EP AC C

18

TE D

17

2

ACCEPTED MANUSCRIPT

1. Introduction

2

Innovation in the food industry is often intertwined with the development of new products with distinct

3

textures using readily-available, cost-effective materials. For the elaboration of novel gel-based foods,

4

however, only a limited number of ingredients are permitted. As it is time-consuming and costly to

5

introduce and approve new functional ingredients (Jones & McClements, 2010), food product developers

6

often prefer to combine existing ingredients to design new structures/texture rather than explore new ones

7

(Champagne & Fustier, 2007).

8

Mixed biopolymers are frequently used in foods as they impart tailor-made textural characteristics

9

(DeMars & Ziegler, 2001; Siegwein, Vodovotz, & Fisher, 2011; Tolstoguzov, 1997). In this regard, a

10

well-studied protein-polysaccharide mixture is that of gelatin and starch wherein the biopolymers remain

11

either as one phase or phase-separate into two phases (Abdulmola, Hember, Richardson, & Morris, 1996;

12

Djakovic, Sovilj, & Milosevic, 1990; Firoozmand, Murray, & Dickinson, 2009; Khomutov, Lashek,

13

Ptitchkina, & Morris, 1995; Marfil, Anhe, & Telis, 2012). Phase separation in this system, which has been

14

ascribed to the thermodynamic incompatibility between these dissimilar biopolymers (Antonov, Grinberg,

15

& Tolstoguzov, 1977; Frith, 2010; Semenova & Dickinson, 2010), results in a variety of morphologies

16

ranging from fractals to droplets and bicontinuous networks (Clark, Richardson, Robinson, Ross-Murphy,

17

& Weaver, 1982; Clark & Ross-Murphy, 1987; Firoozmand, Murray, & Dickinson, 2007; E. R. Morris,

18

1990; V. J. Morris, 1986; Owen & Jones, 1998).

19

Protein-protein interactions also follow diverse pathways, including phase separation (Howell, 1995),

20

precipitation due to electrostatic interactions (Howell, Yeboah, & Lewis, 1995) and/or synergistic

21

interactions (Ngarize, Adams, & Howell, 2004), as has been reported in many mixed gels based on animal

22

and plant-based proteins (Badii & Howell, 2006; Chronakis & Kasapis, 1993; Herrnansson, Altskar, &

23

Jordansson, 1998; Howell & Lawrie, 1984; Leksrisompong & Foegeding, 2011; Pang, Deeth, Sopade,

24

Sharma, & Bansal, 2014; Sengupta & Damodaran, 2000; Walkenström & Hermansson, 1994). Depending

25

on their composition as well as temperature history, a variety of morphologies including droplet-type,

26

particulate or fine-stranded networks can occur in such gels.

27

Tailoring the magnitude of thermodynamic incompatibility between proteins and polysaccharides by

28

using readily-available plant proteins provides a vast opportunity to create phase-separated systems with

29

diverse microstructures and rheological properties. In this regard, the present work explored the ability of

30

solubilized wheat protein isolate (SWPI) or soy protein isolate (SPI) to alter the microstructure and

31

rheological properties of gelatin and gelatin + starch composite gels. The properties of these four

32

biopolymers have been extensively characterized (Ashogbon & Akintayo, 2014; Bietz & Rothfus, 1970;

AC C

EP

TE D

M AN U

SC

RI PT

1

3

ACCEPTED MANUSCRIPT

Liu, 1997; Malhotra & Coupland, 2004; Nesterenko, Alric, Silvestre, & Durrieu, 2013; Patil, Baczynski,

2

& McCurry, 2006; Puppo & Anon, 1998; Singh, Kumar, Sabapathy, & Bawa, 2008; Veraverbeke &

3

Delcour, 2002). Results from this study demonstrated that plant-based proteins such as SPI and SWPI can

4

substantially alter the microstructure and rheological properties of gelatin and gelatin + starch gels.

5

2. Materials and methods

6

2.1. Materials

7

Food-grade gelatin from acid-treated porcine skin (Type A, pI ~7.0-9.0, Bloom 300 ± 25) was purchased

8

from Sigma-Aldrich (Oakville, ON, Canada). Commercial hydroxypropyl tapioca starch (N-DULGE™

9

C1, [E#1440] was kindly supplied by Ingredion Incorporated (Bridgewater, NJ, USA). This weakly-

10

gelling starch is obtained by chemical treatment of native tapioca starch with propylene oxide.

11

Commercial SPI (Pro-Fam® 974, pI ~ 4.5 - 5.1) and commercial SWPI (Prolite® 100, pI ~ 6.5) were

12

kindly supplied by Archer Daniels Midlands Co (Decatur, IL, USA). FITC (fluorescein-5-isothiocyanate,

13

90% HLPC grade) was purchased from Sigma-Aldrich (Oakville, ON, Canada). Rhodamine B was

14

purchased from Acros Organics (New Jersey, USA). Distilled water was used throughout.

15

2.2. Preparation of solutions

16

All biopolymer solutions were prepared in 20 ml sealed screw-cap glass vials and placed in a 90 °C

17

waterbath where they were held for 3 min (gelatin) or 10 min (starch and plant-based protein), with

18

vortexing at 30 s intervals (Fisher Scientific, Nepean, ON, Canada) to promote solubilization. For the

19

preparation of the two-component samples, solutions of twice the required final biopolymer concentration

20

were prepared and equal weights of both biopolymer solutions were mixed. For preparation of the three-

21

component samples, starch and gelatin solutions were prepared separately with 4 times the required final

22

concentration and mixed at equal weights. The mixed solution of gelatin + starch was then mixed with an

23

equal weight of the plant-based protein at twice the required final concentration. For example, for the

24

protein−protein blends, equal weights of a 12 wt% gelatin solution and a 12, 8 or 4 wt% plant-based

25

protein solution were combined to obtain mixtures with 6 wt% gelatin and 6, 4 or 2 wt% plant-based

26

protein. To produce the 3-component blends, initially, equal weight of 24 wt% starch and 24 wt% gelatin

27

were mixed yielding a 12 wt% starch + 12 wt% gelatin blend. Equal amounts of this mixture and a 12, 8

28

or 4 wt% plant-based protein solution were combined to obtain blends of 6 wt% gelatin + 6wt% starch +

29

6, 4 or 2 wt% plant-based protein. For each mixing step, the blends were mixed at ~ 70 °C and again

30

placed in a waterbath at ~ 90 °C for 2-3 min with repeated vortexing. This was followed by

AC C

EP

TE D

M AN U

SC

RI PT

1

4

ACCEPTED MANUSCRIPT

ultrasonication for ~ 10 s at ~ 90 °C to remove any air bubbles (Eumax® UD50SH-2L ultrasonic bath,

2

Kwun Wah Int. Ltd, Hong Kong).

3

2.3. Thermal treatment and small-deformation rheometry

4

Small-deformation rheometry in oscillatory mode and strain sweep tests at a constant frequency (1 Hz)

5

were performed with a Physica MCR 301 rheometer (Anton Paar GmbH, Graz, Austria) equipped with a

6

Peltier plate temperature control unit (P-PTD 200). A parallel plate measuring geometry (PP 25/TG) with

7

a diameter of 25mm was used. The time-dependent storage (G′) and loss (G") moduli were measured at

8

25 °C at a constant frequency of 1 Hz and a target strain of 0.2% for up to 70 min followed by strain

9

sweep tests. To avoid sample drying, the measuring geometry was covered with a solvent trap containing

10

a moist strip of tissue paper. Preceding each measurement, the temperature of the Peltier plate was set at

11

80 °C and the mixed hot biopolymer solution (at 90 °C) was poured directly onto the hot plate. The

12

geometry was then lowered onto the sample to an operating gap width (1 mm), the sample was carefully

13

trimmed and the temperature of the rheometer cell was reduced from 80 °C to 40 °C at 16 °C min-1. The

14

sample was then held at 40 °C for either 30 s or for 10 min. This thermal processing protocol was adopted

15

from our previous work shown to yield different extents of phase separation in mixture of

16

thermodynamically-incompatible biopolymer solutions (Firoozmand & Rousseau, 2014). Using the

17

following thermal treatment: Th = 40 °C and th = 30 s (Th = heating temperature and th = holding time), a

18

group of phase-separated samples with a relatively low extent of phase separation was produced. Using a

19

longer treatment (Th = 40 °C for th = 10 min), another group of samples with a higher extent of phase

20

separation was produced. After these treatments, the temperature of the rheometer cell was reduced from

21

40 °C to 25 °C at 16 °C min-1. After a 2 min hold time at 25 °C, measurements were commenced and

22

lasted 1 hr. With this protocol, the conventional gel state condition (G′ > G′′) was readily satisfied for all

23

samples. All results herein reported are based on at least triplicate runs. The resulting standard deviation

24

for each data point was calculated using OriginPro software and incorporated as error bars in the

25

corresponding graphs.

26

2.4. Confocal laser scanning microscopy

27

Confocal laser scanning microscopy (CLSM) was performed using an upright Zeiss LSM 510 (Carl Zeiss,

28

Toronto, ON, Canada). Solutions of gelatin with ~ 0.001 wt% FITC and plant-based proteins containing

29

~0.0001 wt% Rhodamine B were used, with the former showing a strong affinity for gelatin and the latter

30

with SPI or SWPI. However, in order to compensate for the loss of fluorescence observed during the

31

thermal treatments, a high CLSM laser intensity was used, resulting in some image bleaching. The CLSM

32

was operated in fluorescent mode with an Ar laser source (488 nm) and HeNe1 laser source (543 nm).

AC C

EP

TE D

M AN U

SC

RI PT

1

5

ACCEPTED MANUSCRIPT

The emission spectra were collected with 2 channels set at 505 nm for gelatin (FITC) and 560 nm

2

(Rhodamine B) for the plant-based proteins. To subject the CLSM samples to a thermal treatment similar

3

to the rheology experiments, a small metallic cup (diam = 10 mm, depth = 5 mm) with high thermal

4

conductivity was used as sample holder. This holder was placed on a Linkam PE120 heating/cooling

5

stage equipped with a T95 LinkPad temperature control unit, equilibrated, and then the samples were

6

processed as per the thermal conditioning used for the rheological measurements. A 10 × objective lens

7

(NA: 0.3) and 10 × oculars were used (magnification 100 ×). Images were recorded at 25 °C at a

8

resolution of 1024 × 1024 pixels. Image optimization was performed using the LSM 510’s built-in image

9

analysis software. Images shown herein are representative of the microstructure seen for a given

RI PT

1

composition.

11

2.5. Zeta potential and pH measurements

12

The zeta (ζ)-potential of all single-component biopolymer solutions at 0.5 wt% was measured with a 90

13

Plus particle size analyzer [Brookhaven Instruments Corporation (Holtsville, USA)]. For the pH

14

measurements, each single, two and three-component biopolymer mixture was prepared as per section

15

2.2. Two replicates of each were prepared and the pH was measured five times per replicate using a

16

Fisherbrand Accumet pH meter. The pH results were averaged and reported with the accompanying

17

standard deviation. All solutions were prepared at their natural pH, without any pH adjustment.

18

3.0 Results

19

3.1. Gel microstructure

20

Figure 1A-C shows the microstructure of 6 wt% gelatin mixed with 2-6 wt% SPI. When subjected to Th =

21

40 °C and th = 30 s, all samples exhibited relatively uniform microstructures with no obvious sign of

22

thermally-induced segregation. However, insoluble or unmixed SPI residues were present, with these

23

increasing in size and frequency with concentration. Samples containing 4 and 6 wt% SPI showed the

24

presence of possible coacervates, suggesting a concentration-dependent relationship (Figure 1B and 1C

25

frames). There were no changes in gelatin-SPI mixture microstructure with the longer treatment (Th = 40

26

°C, th = 10 min).

27

Figure 2A-C shows the microstructure of 6 wt% gelatin mixed with 2-6 wt% SWPI. With the shorter

28

thermal treatment, all samples contained numerous clustered SWPI aggregates surrounded by the

29

continuous gelatin phase. The size of the individual spherical elements within the clusters grew in

30

diameter from ~ 5 µm at 2 wt% (Figure 2A) to upwards of 20 µm at 6 wt% wheat protein, demonstrating

AC C

EP

TE D

M AN U

SC

10

6

ACCEPTED MANUSCRIPT

a concentration-dependent effect (Figure 2C). With th = 10 min, the microstructure of the gelatin−SWPI

2

gels did not appreciably change.

3

The 6 wt% gelatin + 6 wt% starch gels subjected to phase separation for 30 s (Figure 3A) or 10 min

4

(Figure 3B) exhibited similar phase-separated microstructures, with greater coarsening in the latter based

5

on the reduced interconnectivity of the gelatin-rich domains (bright region) and greater proportion of the

6

starch-rich domains (dark region) visible.

7

All gelatin + SPI + starch gels exhibited phase-separated microstructures, with the gelatin and SPI

8

remaining mixed as the continuous phase and starch existing as the discontinuous phase with 2 and 4 wt%

9

SPI (Figures 4A and B). The presence of 6 wt% SPI resulted in a complex microstructure where the

10

droplet-type morphology at lower soy protein content was not evident (Figure 4C). Rather, there appeared

11

to be segregation of the gelatin and SPI, implying the possibility of a 3-phase multi-continuous

12

microstructure. The microstructures of these 3 samples did not differ when subjected to hold times of 30 s

13

or 10 min.

14

Figures 5A and B show the microstructure of gelatin + SWPI + starch gels. At 30 s, all samples exhibited

15

multiple phase-separated microstructures wherein the three biopolymers were clearly distinct (Figure

16

5A1-A3). With 2 wt% SWPI (Figure 5A-1), the starch and gelatin formed semi-continuous domains

17

whereas the SWPI aggregates were assembled at the gelatin-starch phase interface (square in Figure 5A-

18

1) and resembled a form of Pickering stabilization. By increasing SWPI to 4 wt% (Figure 5A-2), SWPI

19

aggregates increased in number and volume fraction, which accompanied a visible reduction in the

20

domain size of both the gelatin and starch domains. With 6 wt% SWPI (Figure 5A-3), as a consequence

21

of higher space occupancy by the SWPI, the gelatin and more so the starch phase became more visually

22

discrete from a microstructural perspective. With the longer hold time, the gelatin phase coarsened,

23

particularly at 2 wt % SWPI (Figure 5B-1– see frame). Similar microstructures to the 30 s hold time were

24

evident at 4 wt% and 6 wt% SWPI (Figures 5B-2 and 5B-3), though the gelatin domains were somewhat

25

more coarsened (see frames).

26

3.2. Rheological measurements

27

3.2.1. Small-deformation oscillatory measurements

28

Figure 6 shows the time-dependent elastic (storage) (G′) and viscous (loss) (G″) moduli of the gelatin +

29

plant protein gels. With a hold time of 30 s, the gelatin-only sample showed the lowest G′ after 1 hour

30

(425 ± 10 Pa) and G″ (10 ± 0 Pa). With 2 wt% SWPI, G′ increased to 462 ± 5 Pa whereas addition of 4

31

wt% and 6 wt% SWPI, G′ increased to 545 ± 12 Pa and 663 ± 9 Pa, respectively. The G″ of the gelatin-

AC C

EP

TE D

M AN U

SC

RI PT

1

7

ACCEPTED MANUSCRIPT

only gels increased to 16 ± 1 Pa with 2 wt% SWPI and to 31 ± 1 Pa and 59 ± 1 Pa with 4 and 6 wt%

2

SWPI, respectively, representing large increases of 59%, 202% and 475%, respectively over the control

3

gelatin gel (Figure 6A).

4

Figure 6B shows the time-dependent G′ and G″ of gelatin-SPI mixed gels. With 2 wt% soy protein, G′

5

increased to 647 ± 17 Pa, and to 738 ± 15 Pa and 1047 ± 15 Pa with 4 wt% and 6 wt% SPI, respectively,

6

which represented increases in G′ of 52%, 74% and 146% - a significant rise compared to the wheat-

7

containing gels. Gel G″ increased to 35 ± 1 Pa with 2 wt% SPI, and to 46 ±1 Pa and 65 ± 2 with 4 wt%

8

and 6 wt% SPI – increases of 241%, 345% and 536% vs. the gelatin control.

9

Increasing the hold time to 10 min did not alter the gelatin control G′ (426 ± 3 Pa) and G″ (11 ± 1 Pa)

10

(Figure 6C). Though the addition of 2 wt% SWPI resulted in an identical G′ (427 ± 6 Pa) to the control,

11

presence of 4 wt% and 6 wt% SWPI slightly increased G′ to 496 ± 4 Pa and 665 ± 6 Pa. G′ increased to

12

619 ± 12 Pa, 678 ± 11 Pa and 1157 ± 21 Pa with 2, 4 and 6 wt% SPI, corresponding to increases of 45%,

13

59% and 172%, respectively (Figure 6D). Gel G″ in the presence of SWPI or SPI significantly rose. For

14

example, G″ rose to 56 ± 2 Pa with 6 wt% SWPI – a 448% increase. With 6 wt% SPI, G″ increased to 71

15

± 1 Pa, corresponding to an increase of 548% over the control.

16

Figure 7 shows the time-dependent G′ of the gelatin–starch gels with either SWPI or SPI following a hold

17

time of 30 s or 10 min at 40 °C. After 1 hour, the gelatin–starch control consisted of G′ and G″ values of

18

766 ± 13 Pa and 38 ± 3 Pa, respectively. Addition of 2, 4 or 6 wt% SWPI reduced G′ to 701 ± 21 Pa, 614

19

± 10 Pa and 485 ± 12 Pa, respectively (Figure 7A). By contrast, G′ increased to 1397 ± 25 Pa, 1650 ± 43

20

Pa and 1050 ± 17 Pa with 2, 4 or 6 wt% SPI, respectively (Figure 7B). Unusually, the change in G′ did

21

not follow the expected trend with increases of 82 % and 115 % with 2 wt% and 4 wt% SPI but only 37%

22

with 6 wt% added SPI. The G″ of the gelatin-starch gels containing SWPI and SPI also rose. With 2, 4

23

and 6 wt% SWPI, G″ increased to 64 ± 5 Pa, 105 ± 8.1 Pa and 152 ± 5.6 Pa corresponding to increases of

24

69%, 176% and 299%, respectively. Similarly, with 2, 4 and 6 wt% SPI, G″ grew to 79 ± 1 Pa, 114 ± 2 Pa

25

and 143 ± 10 Pa – increases of 108%, 200% and 275%, respectively.

26

Increasing the hold time of these composites to 10 min resulted in different trends. Expectedly, the

27

gelatin–starch control showed a lower G′ and G″ compared to the 30 s hold time (381 ± 15 Pa and 40 ± 1

28

Pa, respectively). With the wheat protein, G′ increased to 525 ± 8 Pa with 2 wt% SWPI, with smaller

29

changes observed at 4 wt% (471 ± 9 Pa) and 6 wt% SWPI (425 ± 2 Pa) (Figure 7C). With the soy protein,

30

G′ rose to 1327 ± 21 Pa and 1467 ± 64 Pa with 2 wt% and 4 wt% SPI, but only to 871 ± 49 Pa with 6 wt%

31

SPI (Figure 7D). G″ increased in the presence of SWPI and SPI. For example, with 6wt% SWPI, G″ rose

AC C

EP

TE D

M AN U

SC

RI PT

1

8

ACCEPTED MANUSCRIPT

to 148 ± 5 Pa, an increase of 269 % whereas with 6 wt% SPI, it only rose to 71 ± 1 Pa – an increase of 75

2

% over the control.

3

3.2.2. Strain sweep tests at a constant frequency

4

Figures 8 and 9 show strain sweep tests at constant frequency (1 Hz) and temperature (25 °C) on samples

5

subjected to Th = 40 °C for th = 10 min. In these tests, changes in complex shear modulus (G*) as a

6

function of shear strain amplitude in the oscillatory mode were monitored. Increasing the strain (γ)

7

resulted in macroscopic gel fracture, causing a sharp decrease in G*. Before reaching the fracture point,

8

most samples showed strain-hardening (Groot, Bot, & Agterof, 1996) whereby G* increased with the

9

magnitude of γ and the gels became resistant to flow under greater deformation. The % strain hardening

SC

RI PT

1

10

(% sh) was defined as follows:

11

% ℎ =

12

∗ ∗ where  is the maximum G* and  is the initial G*.

13

The gelatin gel showed the maximum % sh (87%) at γ = 3.2. Addition of SWPI reduced both % sh and γ

14

values (61 % and 47 % at γ = 2.7 with 2 and 4 wt% SWPI; 44 %, γ = 1.2 at 6 wt% SWPI) (Figure 8A).

15

Changes in % sh were not as straightforward upon addition of SPI (Figure 8B). By adding 2 and 4 wt%

16

SPI, % sh decreased to 51 % and 55 % (both at γ = 2.7) but only diminished to 32 % at γ = 2.3 with 6

17

wt% SPI.

18

Presence of starch in the gelatin-plant protein composites radically altered % sh behaviour (Figure 9A). In

19

the gelatin-starch control, % sh plummeted to 23 % at γ = 1.6 compared to the gelatin-only gel. With 2

20

wt% SWPI, only minor strain hardening was apparent whereas at 4 wt%, slight strain softening was

21

evident prior to the gel yielding. With 6 wt% SWPI, G* value diminished with strain demonstrating gel

22

flowability. With 2 and 4 wt% SPI, % sh values were 14 % and 26 % at γ = 1.6 and at γ = 2.2,

23

respectively (Figure 9B). No strain hardening was evident with addition of 6 wt% SPI.

24

4. Discussion

25

4.1. Microstructure

26

The intent of this study was to extend two-component phase separation via addition of a potentially

27

incompatible third component. In so doing, we wanted to widen the range of possible microstructures and

28

rheological properties attainable using standard gelatin-starch biopolymer mixtures. Some of the inherent

29

properties of the plant proteins impacted the observed phase separation behaviour. At the pH values

TE D

M AN U

× 100 (1)

AC C

EP

∗ ∗    ∗  

9

ACCEPTED MANUSCRIPT

investigated, the ζ-potential of gelatin was 10.8 ± 0.3 mV whereas those of the SWPI and SPI were 14.1 ±

2

0.4 mV and −39.2 ± 1.7 mV, respectively. In samples containing gelatin plus SWPI, the pH was below

3

the pI of both proteins [gelatin (7-9) and SWPI (~6.5)] (Table 1). Given their positive ζ-potential values,

4

these proteins repelled one another leading to SWPI aggregate formation and protein-protein phase

5

separation. In samples containing gelatin and SPI, the pH values were above the pI of the SPI (4.5-5.1)

6

and below that of the gelatin (7-9). With its negative ζ-potential, SPI interacted with the positively-

7

charged gelatin polypeptide chains and remained as one phase, thereby demonstrating greater

8

compatibility than the wheat protein.

9

Polypeptide folding (Tolstoguzov, 1999) and space limitations caused by the protein’s excluded volume

10

(Tolstoguzov, 1997) may have enhanced the association of like biopolymers in the thermodynamically

11

incompatible mixture of gelatin and SWPI, resulting in concurrent gelatin-wheat protein segregation and

12

self-aggregation of the SWPI within the continuous gelatin phase. A factor likely influencing SPI-gelatin

13

compatibility was the existence of glucoside units in the former (Kimura, et al., 2008), as these drive the

14

hydrophobic–hydrophilic balance toward greater hydrophilicity. As protein incompatibility is strongly

15

influenced by difference in hydrophobicity (Polyakov, et al., 1997), the soy protein’s greater

16

hydrophilicity likely promoted its compatibility with gelatin. As both plant proteins were thermally-

17

denatured due to heating at 95 °C during sample preparation, this likely exposed buried hydrophobic

18

domains which further altered their interactions with the gelatin (Gosal & Ross-Murphy, 2000; Ross-

19

Murphy, 1998). However, this was not investigated in detail.

20

Classic polysaccharide−protein phase separation took place in the gelatin + starch blends, whereby

21

composition and thermal treatment dictated gel rheology and morphology (Tolstoguzov, 2003). However,

22

in the gelatin + SPI + starch gels, segregative interactions between gelatin and starch and associative

23

interactions between gelatin and SPI took place whereas in the gelatin + SWPI + starch gels, multiple

24

segregative interactions between gelatin and starch as well as between gelatin and SWPI occurred. In both

25

cases, the plant-based proteins partitioned towards the most favourable phase - the SWPI, with its positive

26

ζ-potential, repelled the positively-charged gelatin and preferentially partitioned towards the near-neutral

27

starch (ζ-potential:−2.5 ± 1.1 mV). The SPI behaved differently given its negative ζ-potential as it

28

interacted with the positively-charged gelatin phase.

29

4.2. Rheological measurements

30

All protein–protein mixtures exhibited higher G′ values than the gelatin-only gel showing that increasing

31

the total solids content in the mixture increased G′. Figure 10A compares the final G′ of the protein-

AC C

EP

TE D

M AN U

SC

RI PT

1

10

ACCEPTED MANUSCRIPT

protein gels aged one hour aged following Th= 40 °C for th= 30 s or th= 10 min. G′ was generally lower

2

following th= 10 min in both wheat and soy-containing gels, except for the 6 wt% gelatin + 6 wt% wheat

3

protein gel which saw no change as a function of hold time and the 6 wt% gelatin + 6 wt% soy protein gel

4

whose G′ increased with hold time.

5

Alone, gelatin network elasticity arises from small locally ordered crystallites (or junction zones)

6

connected by a mesh of entangled flexible polymers, stabilized predominantly by hydrogen bonding and

7

other secondary forces in a co-operative interchain fashion. With dissimilar components (i.e., gelatin +

8

SWPI), thermally-induced phase separation results in the concentration of similar components into

9

separated domains via uphill diffusion (Nishi, Wang, & Kwei, 1975), which lowers contact between the

10

dissimilar protein molecules due to an excluded volume effect. The reduced extent of contact between

11

dissimilar protein molecules will reduce the possibility of synergistic interactions between them and as a

12

consequence G′ will be reduced with time, e.g., in gels containing 6 wt% gelatin mixed with 2 and 4 wt%

13

wheat or soy protein after 10 min vs. 30 s. However, at a higher plant-based protein concentration (e.g., 6

14

wt%), given that the biopolymer (protein) content increased, uphill diffusion was hindered as a portion of

15

the solvent was absorbed by the protein molecules themselves. Speculatively, the result was that a greater

16

proportion of the dissimilar biopolymers (i.e., proteins) remained in close proximity producing a more

17

interdigitated network and hence higher G′ values.

18

The mixed gelatin-starch-plant protein gels showed radically altered rheological properties (Figure 10B).

19

Presence of SWPI systematically reduced G′ with increasing concentration at longer thermal treatments.

20

As per the ζ-potential measurements and CLSM observations, the SWPI and gelatin chains repelled one

21

another. It is likely that as the SWPI withdrew from the gelatin phase and formed dispersed aggregates, it

22

acted as an inactive filler effectively reducing gelatin-gelatin interactions with a concomitant reduction in

23

G′ (Chen & Dickinson, 1999; van Vliet, 1988). This is clearly evident in samples containing 2-6 wt%

24

SWPI where, following a thermal treatment of 30 s, G″ increased 69 %, 176 % and 299 % with only

25

small corresponding increases in G′ (9 - 37 % only).

26

By contrast, with its negative ζ-potential, SPI partitioned within the gelatin phase thereby behaving as an

27

active filler at 2 and 4 wt% thus increasing G′ (Ring & Stainsby, 1982). At 6 wt% SPI, the high

28

concentration of biopolymer (18 wt%) likely intensified the extent of thermodynamic incompatibility

29

between the biopolymers thus impacting network formation within the gel matrix. Such behaviour has

30

been reported in other systems such as pea protein/kappa-carrageenan/starch gels where multicomponent

31

phase separation reduces G′ (Nunes, Raymundo, & Sousa, 2006).

AC C

EP

TE D

M AN U

SC

RI PT

1

11

ACCEPTED MANUSCRIPT

During temperature-induced phase separation, different factors influence gel rheology and microstructure,

2

including partitioning of water between the phases (Clark, 1987; Kasapis, Morris, Norton, & Clark,

3

1993b), gelation of individual components (Kasapis, Morris, Norton, & Clark, 1993a) and the extent of

4

gelatin cross-linking (Firoozmand, et al., 2009). In the present case, it is highly likely that water

5

partitioning was impacted, as were gelation and physical cross-linking of the gelatin chains. As noted,

6

with being interdigitated within the gelatin, the soy protein reinforced the gelatin’s G′ whereas with wheat

7

protein, that was not the case. Overall, the dynamics of the mixed systems were multifaceted, and with it a

8

complex picture of segregative/associative dynamics emerged.

9

Large deformation rheology provided further insight into the physical properties and flow behaviour of

10

the gels. Figures 8 indicates that the gelatin gel became less elastic in the presence of the plant-based

11

proteins, as the % sh of the gelatin-only gel (86 %) fell to 32-55 % with SPI and 44-61% with SWPI.

12

Hence, addition of both proteins reduced the strain-hardening behaviour seen in gelatin gels, diminishing

13

the ‘extendibility’ of gelatin polypeptide chains presumably at a molecular level due to the interference of

14

gelatin triple helix formation and physical cross-linking (Djabourov, 1988).

15

Starch also reduced gelatin’s strain-hardening behaviour, e.g., upon addition of 6 wt% starch to the

16

gelatin gel % sh dropped from 86% to 23% (Figure 9). The presence of starch in protein–protein mixtures

17

altered the flow behaviour of the gels significantly. Values of % sh showed negative values with 2-4 wt%

18

SWPI whereas with 6 wt% SWPI the gel was flowable, with no evidence of strain-hardening. This was

19

likely due to the presence of dispersed domains containing incompatible biopolymers as observed by

20

others in mixed gels (Zasypkin, Braudo, & Tolstoguzov, 1997). Finally, the gelatin + soy + starch gels

21

showed slight changes in % sh (< 25 %) with 2-4 wt% SPI whereas at 6 wt% SPI, strain hardening was

22

not measurable indicative of complete gel flowability. At this concentration, gel G′ decreased as the

23

elasticity of the gelatin network was reduced and consequently the gel network could not resist

24

deformation thereby flowing under increased strain.

25

5. Conclusions

26

The impact of two plant-based proteins, SPI and SWPI, on the microstructure and rheology of protein–

27

polysaccharide composites was explored. The resulting gels revealed striking microstructural and

28

rheological differences, with the ζ-potential of the biopolymers playing an influential role. Notably, with

29

similar ζ-potential values, the SWPI and gelatin repelled one another whereas with SPI, there was

30

interaction with gelatin. In the presence of starch (which was thermodynamically incompatible with

31

gelatin), the wheat protein partitioned as a third phase leading to the formation of a three-phase system

AC C

EP

TE D

M AN U

SC

RI PT

1

12

ACCEPTED MANUSCRIPT

with a common solvent. Such a system may be considered a water-in-water-in-water emulsion. With soy,

2

such segregation was not as evident, given the association of soy with gelatin.

3

Presence of the plant-based proteins in the gelatin and gelatin-starch gels altered viscoelasticity and

4

generally increased gel modulus. The most remarkable rheological property of the mixed gels was their

5

difference in strain-hardening which greatly differed amongst the mixed gels. The development of such

6

strain-softening gels may lead to the development of a new class of gel matrices that impart different

7

sensory attributes, facilitate easier chewing and mastication for children and an elderly population, as well

8

as new food products with soft, extendable textures to replace oil/fat in food matrices. It is clear that such

9

gels command continued study.

SC

RI PT

1

6. Acknowledgements

11

Funding from the Natural Sciences and Engineering Research Council of Canada is greatly appreciated.

AC C

EP

TE D

M AN U

10

13

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

Abdulmola, N. A., Hember, M. W. N., Richardson, R. K., & Morris, E. R. (1996). Application of polymer blending laws to starch-gelatin composites. Carbohydrate Polymers, 31(1-2), 53-63. Antonov, Y. A., Grinberg, V. Y., & Tolstoguzov, V. B. (1977). Phase-equilibria in water-protein polysaccharide systems, II. water-casein-neutral polysaccharide systems. Colloid and Polymer Science, 255(10), 937-947. Ashogbon, A. O., & Akintayo, E. T. (2014). Recent trend in the physical and chemical modification of starches from different botanical sources: A review. Starch-Starke, 66(1-2), 41-57. Badii, F., & Howell, N. K. (2006). Fish gelatin: Structure, gelling properties and interaction with egg albumen proteins. Food Hydrocolloids, 20(5), 630-640. Bietz, J. A., & Rothfus, J. A. (1970). Comparison of peptides from wheat gliadin and glutenin. Cereal Chemistry, 47(4), 381-&. Champagne, C. P., & Fustier, P. (2007). Microencapsulation for the improved delivery of bioactive compounds into foods. Current Opinion in Biotechnology, 18(2), 184-190. Chen, J. S., & Dickinson, E. (1999). Effect of surface character of filler particles on rheology of heat-set whey protein emulsion gels. Colloids and Surfaces B-Biointerfaces, 12(3-6), 373-381. Chronakis, I. S., & Kasapis, S. (1993). Structural properties of single and mixed milk/soya protein systems. Food Hydrocolloids, 7(6), 459-478. Clark, A. H. (1987). The application of network theory to food systems. In J. M. V. Blanshard & P. J. Lillford (Eds.), Food Structure and Behaviour (pp. 13-34). London: Academic Press. Clark, A. H., Richardson, R. K., Robinson, G., Ross-Murphy, S. B., & Weaver, A. C. (1982). Structural and mechanical properties of agar/BSA co-gels. Progress in Food and Nutrition Science, 6(1-6), 149-160. Clark, A. H., & Ross-Murphy, S. B. (1987). Structural and mechanical properties of biopolymer gels. In A. H. Clark, K. Kamide, S. B. Ross-Murphy & M. Saito (Eds.), Advances in Polymer Science, Biopolymers (Vol. 83, pp. 57-192). Heidelberg: Springer-Verlag. DeMars, L. L., & Ziegler, G. R. (2001). Texture and structure of gelatin/pectin-based gummy confections. Food Hydrocolloids, 15(4-6), 643-653. Djabourov, M. (1988). Architecture of gelatin gels. Contemporary Physics, 29(3), 273-297. Djakovic, L., Sovilj, V., & Milosevic, S. (1990). Rheological behavior of thixotropic starch and gelatin gels. Starch-Starke, 42(10), 380-385. Firoozmand, H., Murray, B. S., & Dickinson, E. (2007). Fractal-type particle gel formed from gelatin plus starch solution. Langmuir, 23(8), 4646-4650. Firoozmand, H., Murray, B. S., & Dickinson, E. (2009). Microstructure and rheology of phase-separated gels of gelatin plus oxidized starch. Food Hydrocolloids, 23(4), 1081-1088. Firoozmand, H., & Rousseau, D. (2014). Tailoring the morphology and rheology of phase-separated biopolymer gels using microbial cells as structure modifiers. Food Hydrocolloids, 42, 204-214. Frith, W. J. (2010). Mixed biopolymer aqueous solutions - phase behaviour and rheology. Advances in Colloid and Interface Science, 161(1-2), 48-60. Gosal, W. S., & Ross-Murphy, S. B. (2000). Globular protein gelation. Current Opinion in Colloid & Interface Science, 5(3-4), 188-194. Groot, R. D., Bot, A., & Agterof, W. G. M. (1996). Molecular theory of strain hardening of a polymer gel: Application to gelatin. Journal of Chemical Physics, 104(22), 9202-9219. Herrnansson, A., Altskar, A., & Jordansson, E. (1998). Phase-separated mtxed gelatin-milk protein systems. In G. O. Phillips & P. A. Williams (Eds.), Gums and Stabilisers for the Food Industry 9 (pp. 107-116): The Royal Society of Chemistry. Howell, N. K. (1995). Synergism and interaction in mixed food protein systems. In S. E. Harding, S. E. Harding & J. R. Mitchell (Eds.), Biopolymer Mixtures. (pp. 329-347 ). Nottingham, U.K.: Nottingham University Press.

EP

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

References

AC C

1

14

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Howell, N. K., & Lawrie, R. A. (1984). Functional-aspects of blood-plasma proteins .2. Gelling properties. Journal of Food Technology, 19(3), 289-295. Howell, N. K., Yeboah, N. A., & Lewis, D. F. V. (1995). Studies on the electrostatic interactions of lysozyme with alpha-lactalbumin and beta-lactoglobulin. International Journal of Food Science and Technology, 30(6), 813-824. Jones, O. G., & McClements, D. J. (2010). Functional Biopolymer Particles: Design, Fabrication, and Applications. Comprehensive Reviews in Food Science and Food Safety, 9(4), 374-397. Kasapis, S., Morris, E. R., Norton, I. T., & Clark, A. H. (1993a). Phase-equilibria and gelation in gelatin maltodextrin systems. Part I. gelation of individual components. Carbohydrate Polymers, 21(4), 243-248. Kasapis, S., Morris, E. R., Norton, I. T., & Clark, A. H. (1993b). Phase-equilibria and gelation in gelatin maltodextrin systems. Part IV: composition-dependence of mixed-gel moduli. Carbohydrate Polymers, 21(4), 269-276. Khomutov, L. I., Lashek, N. A., Ptitchkina, N. M., & Morris, E. R. (1995). Temperature-composition phase diagram and gel properties of the gelatin-starch-water system. Carbohydrate Polymers, 28(4), 341-345. Kimura, A., Fukuda, T., Zhang, M., Motoyama, S., Maruyama, N., & Utsumi, S. (2008). Comparison of physicochemical properties of 7s and 11s globulins from pea, fava bean, cowpea, and french bean with those of soybean-french bean 7s globulin exhibits excellent properties. Journal of Agricultural and Food Chemistry, 56(21), 10273-10279. Leksrisompong, P. N., & Foegeding, E. A. (2011). How micro-phase separation alters the heating rate effects on globular protein gelation. Journal of Food Science, 76(3), E318-E327. Liu, K. (1997). Chemistry and Nutritional Value of Soybean Components. In K. Liu (Ed.), Soybeans (pp. 25-113): Springer US. Malhotra, A., & Coupland, J. N. (2004). The effect of surfactants on the solubility, zeta potential, and viscosity of soy protein isolates. Food Hydrocolloids, 18(1), 101-108. Marfil, P. H. M., Anhe, A., & Telis, V. R. N. (2012). Texture and Microstructure of Gelatin/Corn StarchBased Gummy Confections. Food Biophysics, 7(3), 236-243. Morris, E. R. (1990). Mixed Polymer Gel. In P. Harris (Ed.), Food Gels. (pp. 291-359). London, U.K.: Elsevier Applied Science. Morris, V. J. (1986). Multicomponent gels. In G. O. Phillips, P. A. Williams & D. J. Wedlock (Eds.), Gum and Stabilisers for the Food Industry 3 (pp. 87-99). London, U.K.: Elsevier Applied Science. Nesterenko, A., Alric, I., Silvestre, F., & Durrieu, V. (2013). Vegetable proteins in microencapsulation: A review of recent interventions and their effectiveness. Industrial Crops and Products, 42, 469479. Ngarize, S., Adams, A., & Howell, N. K. (2004). Studies on egg albumen and whey protein interactions by FT-Raman spectroscopy and rheology. Food Hydrocolloids, 18(1), 49-59. Nishi, T., Wang, T. T., & Kwei, T. K. (1975). Thermally induced phase separation behavior of compatible polymer mixtures. Macromolecules, 8(2), 227-234. Nunes, M. C., Raymundo, A., & Sousa, I. (2006). Rheological behaviour and microstructure of pea protein/kappa-carrageenan/starch gels with different setting conditions. Food Hydrocolloids, 20(1), 106-113. Owen, A. J., & Jones, R. A. L. (1998). Rheology of a simultaneously phase-separating and gelling biopolymer mixture. Macromolecules, 31(21), 7336-7339. Pang, Z. H., Deeth, H., Sopade, P., Sharma, R., & Bansal, N. (2014). Rheology, texture and microstructure of gelatin gels with and without milk proteins. Food Hydrocolloids, 35, 484-493. Patil, S. K., Baczynski, M., & McCurry, T. (2006). Wheat protein isolates - Alternative to sodium caseinate. Cereal Foods World, 51(5), 279-281. Polyakov, V. I., Grinberg, V. Y., & Tolstoguzov, V. B. (1997). Thermodynamic incompatibility of proteins. Food Hydrocolloids, 11(2), 171-180.

AC C

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

15

ACCEPTED MANUSCRIPT

33

RI PT

SC

M AN U

TE D

EP

32

Puppo, M. C., & Anon, M. C. (1998). Structural properties of heat-induced soy protein gels as affected by ionic strength and pH. Journal of Agricultural and Food Chemistry, 46(9), 3583-3589. Ring, S., & Stainsby, G. (1982). Filler reinforcement of gels. Progress in Food and Nutrition Science, 6(1-6), 323-329. Ross-Murphy, S. B. (1998). Reversible and irreversible biopolymer gels - Structure and mechanical properties. Berichte Der Bunsen-Gesellschaft-Physical Chemistry Chemical Physics, 102(11), 1534-1539. Semenova, M. G., & Dickinson, E. (2010). Biopolymers in food colloids: thermodynamics and molecular interactions. Leiden, Netherlands: Brill Academic PUB. Sengupta, T., & Damodaran, S. (2000). Incompatibility and phase separation in a bovine serum albumin/β-casein/water ternary film at the air–water Interface. Journal of Colloid and Interface Science, 229(1), 21-28. Siegwein, A. M., Vodovotz, Y., & Fisher, E. L. (2011). Concentration of Soy Protein Isolate Affects Starch-Based Confections' Texture, Sensory, and Storage Properties. Journal of Food Science, 76(6), E422-E428. Singh, P., Kumar, R., Sabapathy, S. N., & Bawa, A. S. (2008). Functional and edible uses of soy protein products. Comprehensive Reviews in Food Science and Food Safety, 7(1), 14-28. Tolstoguzov, V. (1997). Thermodynamic aspects of dough formation and functionality. Food Hydrocolloids, 11(2), 181-193. Tolstoguzov, V. (1999). Origins of globular structure in proteins. Febs Letters, 444(2-3), 145-148. Tolstoguzov, V. (2003). Thermodynamic considerations of starch functionality in foods. Carbohydrate Polymers, 51(1), 99-111. van Vliet, T. (1988). Rheological properties of filled gels - influence of filler matrix interaction. Colloid and Polymer Science, 266(6), 518-524. Veraverbeke, W. S., & Delcour, J. A. (2002). Wheat protein composition and properties of wheat glutenin in relation to breadmaking functionality. Critical Reviews in Food Science and Nutrition, 42(3), 179-208. Walkenström, P., & Hermansson, A.-M. (1994). Mixed gels of fine-stranded and particulate networks of gelatin and whey proteins. Food Hydrocolloids, 8(6), 589-607. Zasypkin, D. V., Braudo, E. E., & Tolstoguzov, V. B. (1997). Multicomponent biopolymer gels. Food Hydrocolloids, 11(2), 159-170.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

16

ACCEPTED MANUSCRIPT

Table legend:

AC C

EP

TE D

M AN U

SC

RI PT

Table 1: The pH values of biopolymer solutions, St: starch; Ge: gelatin; SWPI: soluble wheat protein isolate; SPI: soy protein isolate.

ACCEPTED MANUSCRIPT

SC

TE D EP AC C

pH 4.82 ± 0.02 3.97 ± 0.02 4.01 ± 0.12 3.98 ± 0.12 4.72 ± 0.05 4.66 ± 0.04 4.63 ± 0.02 4.6 ± 0.01 4.5 ± 0.03 4.5 ± 0.07

RI PT

pH Sample 4.81 ± 0.08 6 wt% G + 6 wt% St 6.06 ± 0.21 7.17 ± 0.01 2 wt% SWPI 7.04 ± 0.02 4 wt% SWPI 6.98 ± 0.02 6 wt% SWPI 5.62 ± 0.02 6 wt% G + 2 wt% SWPI 5.98 ± 0.02 6 wt% G + 4 wt% SWPI 6.15 ± 0.02 6 wt% G + 6 wt% SWPI 5.46 ± 0.02 6 wt% St + 6wt% G + 2 wt% SWPI 5.86 ± 0.03 6 wt% St + 6wt% G + 4 wt% SWPI 6.04 ± 0.01 6 wt% St + 6wt% G + 6 wt% SWPI

M AN U

Sample 6 wt% G 6 wt% St 2 wt% SPI 4 wt% SPI 6 wt% SPI 6 wt% G + 2 wt% SPI 6 wt% G + 4 wt% SPI 6 wt% G + 6 wt% SPI 6 wt% St + 6wt% G + 2 wt% SPI 6 wt% St + 6wt% G + 4 wt% SPI 6 wt% St + 6wt% G + 6 wt% SPI

ACCEPTED MANUSCRIPT

Figure legend: Figure 1: CLSM of gels made of 6 wt % gelatin mixed with 2 wt % (A) 4 wt % (B) or 6 wt% (C) SPI subjected to Th = 40 °C and th = 30 s. Rounded squares show areas of possible SWPI coacervation.

RI PT

Figure 2: CLSM of gels made of 6 wt % gelatin mixed with 2 wt % (A) 4 wt % (B) or 6 wt% (C) SWPI subjected to (A) Th = 40 °C and th = 30 s. The bright regions represent clusters of aggregated SWPI. Figure 3: CLSM of gels made of 6 wt % gelatin mixed with 6 wt % starch subjected to Th = 40 °C and th = 30 s (A) or th = 10 min (B). The bright regions are gelatin-rich whereas the dark regions are maltodextrin-

SC

rich. The same region is shown in both images confirming the increased extent of coarsening with time. Figure 4: CLSM of gels made of 6 wt % gelatin and 6 wt % starch containing 2 wt % (A) 4 wt % (B) or 6

M AN U

wt% (C) SPI subjected to Th = 40 °C and th = 30 s. The bright regions are gelatin- and soy-rich regions and the dark regions are starch-rich.

Figure 5: CLSM of gels made of 6 wt % gelatin and 6 wt % starch containing SWPI subjected to Th = 40 °C and th = 30 s (A) or th = 10 min (B). A-1 & B-1: 2 wt % SWPI; A-2 & B-2: 4 wt % SWPI; A-3 & B-3: 6 wt % SWPI. The dark grey (red in coloured version) patch-like regions are gelatin-rich whereas the white (yellow-green in coloured version) droplet-like features are wheat-rich regions and the dark regions

TE D

are starch-rich. Rounded squares show areas of interest (see text for details). Figure 6: Time-dependent changes in G′ (filled symbols) and G" (open symbols) at 25 °C for gels subjected to Th = 40 °C and th = 30 s [(A) and (B)] or Th = 40 °C and th = 10 min [(C) and (D)]. Left-hand column – SWPI; Right-hand column – SPI. Gel made with 6 wt % gelatin ,

% SPI, , .

; 4 wt % SWPI

,

EP

wt % SWPI

; 6 wt % SWPI

,

, ; 6 wt% gelatin gel and: 2

; 2 wt % SPI

,

; 4 wt % SPI

, ; 6 wt

AC C

Figure 7: Time-dependent changes in G′ (filled symbols) and G" (open symbols) at 25 °C for gels subjected to Th = 40 °C and th = 30 s [(A) and (B)] or th = 10 min [(C) and (D)]. Gel made with 6 wt % gelatin+ 6 wt% starch wt % SWPI

,

, ; gels made with 6 wt % gelatin+ 6 wt% starch and: 2 wt % SWPI

; 6 wt % SWPI

,

; 2 wt % SPI

,

; 4 wt % SPI

,

;4

, ; 6 wt % SPI, , .

Figure 8: Strain-dependent complex modulus (G*) of gelatin and gelatin-plant protein gels subjected to Th = 40 °C and th = 10 min after ageing 1 hour at 25 °C. 6 wt % gelatin gel -‘0’; 6wt% gelatin gel + plant-based protein at: 2 wt% -‘2’; 4 wt% - ‘4’; 6 wt% - ‘6’. (A) SWPI; (B) SPI. Note different y-axis scales in A and B.

ACCEPTED MANUSCRIPT

Figure 9: Strain-dependent complex modulus (G*) of gelatin-starch and gelatin-starch-plant protein gels subjected to Th = 40 °C and th = 10 min after ageing 1 hour at 25 °C. 6 wt % gelatin + 6 wt% starch gel ‘0’; 6 wt% gelatin-starch gel + plant-based protein at: 2 wt% -‘2’; 4 wt% - ‘4’; 6 wt% - ‘6’. (A) SWPI; (B) SPI. Note different y-axis scales in A and B.

RI PT

Figure 10: (A) G′ of 6 wt % gelatin gels alone and mixed with plant-based proteins; (B) G′ of 6 wt% gelatin + 6 wt% starch gels alone and mixed with 2-6 wt% plant-based proteins. All samples subjected to Th = 40 °C for th = 30 s ( ) or th = 10 min (

) after ageing 1 hour at 25 °C. G: gelatin; St: starch; W:

AC C

EP

TE D

M AN U

SC

wheat protein; S: Soy protein.

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

Figure 1

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

Figure 2

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

Figure 3

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

Figure 4

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 5

ACCEPTED MANUSCRIPT

s

100

10

10

RI PT

100

Time (min) 0

10

20

30

40

50

60

0

Figure 6A

SC

G' & G" (Pa)

1000

G' & G" (Pa)

1000

10

20

30

Time (min)

40

50

60

Figure 6B

1000

G' & G" (Pa)

G' & G" (Pa)

M AN U

1000

100

100

10

TE D

10

Time (min)

10

30

AC C

Figure 6C

20

40

EP

0

50

60

Time (min) 0

10

20

30

Figure 6D

40

50

60

ACCEPTED MANUSCRIPT

G' & G" (Pa) 100

Time (min)

10 0

10

20

30

40

50

10

60

0

Figure 7A

10

SC

G' & G" (Pa) 100

RI PT

1000

1000

20

30

Time (min)

40

50

60

40

50

Figure 7B

1000 G' & G" (Pa)

G' & G" (Pa)

M AN U

1000

100

TE D

100

Time (min)

10 0

10

30

40

AC C

EP

Figure 7C

20

50

60

Time (min)

10 0

10

20

Figure 7D

30

60

ACCEPTED MANUSCRIPT

1100 1000

6 6 6 6

800 66 66 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

700

66

6

6

4

44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

500

444

0 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 22 02 02 00 2 02

0 2

44 0 02 2

4 0 2

4

0 0 44 40 2 6 0 4 02 2 0 2

4

0 2 0 2

2 6

SC

600

6

400

2

4

1

10

Strain γ (%) 100

Figure 8A 1600

6 66

TE D

1400

66 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

1200

6

1000

6

6

66

6

44 42 2 42 4 6 4 0 4 2 4 2 02 4422 4 4 4 4 0 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 22 0 22 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 0 00 0 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1000

EP

800

AC C

600

G* (Pa)

6

M AN U

G* (Pa)

4

300

400

RI PT

900

1

Figure 8B

10

6 0 6 4 2

6 4 2 6 4

Strain γ (%) 100

6

1000

ACCEPTED MANUSCRIPT

600

500

44 4 4 4 4 4 4 4 4 4 4

444

22222

2 22

222 2

RI PT

22 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

0 0 0 4444444442 0 66 6 6 6 6 6 6 6 6 6 6 666 4 66 0 66 0 66 4 0 66 06 0 6 0 6 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 6 6 04 6

SC

44

M AN U

G* (Pa)

400

444

6

300 1

Strain γ (%) 100

10

Figure 9A 2000 1800 1600 1400

TE D

1000

66 6 6 6 6 6 6 6 6 6 6 6 6 6

800

6666

EP

600

666

666

00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AC C

400

G* (Pa)

1000

44 4 4 4 44 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 22 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

1200

200

2

1

Figure 9B

10

66

66 2 6 6

00

0

4

0 00 0 6 0

6

Strain γ (%) 100

2

4 6

4 6 4

1000

ACCEPTED MANUSCRIPT

1200 1000

RI PT

800

SC

G' (Pa)

600

6G

6G2W

6G4W

6G6W

Figure 10A 1600 1400

1000 800

AC C

400

G' (Pa)

6G4S

6G6S

EP

600

200

6G2S

TE D

1200

M AN U

400

t 2S 4S 6S 4W 6W 6S 2W St+ St+ St+ 6G St+ St+ St+ 6 6 6 6 6 6 G G G 6 6 6 6G 6G 6G

Figure 10B

ACCEPTED MANUSCRIPT

1 2 3 4 5

Highlights • • • •

Protein-protein-polysaccharide biopolymer solutions to diversify gel microstructure and rheology Creation of water-in-water-in-water emulsions with protein−protein−polysaccharide solutions Zeta potential impacts the phase behaviour of biopolymer solutions Change in rheology of starch−gelatin gel with addition of plant-based proteins

AC C

EP

TE D

M AN U

SC

RI PT

6 7

1