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j.foodhyd.2018.08.026

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Accepted Manuscript
Linking rheology and printability of a multicomponent gel system of carrageenanxanthan-starch in extrusion based additive manufacturing
Zhenbin Liu, Bhesh Bhandari, Sangeeta Prakash, Sylvester Mantihal, Min Zhang
PII:
S0268-005X(18)31168-8
DOI:
10.1016/j.foodhyd.2018.08.026
Reference:
FOOHYD 4607
To appear in:
Food Hydrocolloids
Received Date:
27 June 2018
Accepted Date:
15 August 2018
Please cite this article as: Zhenbin Liu, Bhesh Bhandari, Sangeeta Prakash, Sylvester Mantihal,
Min Zhang, Linking rheology and printability of a multicomponent gel system of carrageenanxanthan-starch in extrusion based additive manufacturing, Food Hydrocolloids (2018), doi: 10.1016
/j.foodhyd.2018.08.026
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ACCEPTED MANUSCRIPT
Linking rheology and printability of a multicomponent gel system of
carrageenan-xanthan-starch in extrusion based additive manufacturing
Graphical abstract
ACCEPTED MANUSCRIPT
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Linking rheology and printability of a multicomponent gel system of
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carrageenan-xanthan-starch in extrusion based additive manufacturing
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Zhenbin Liua, Bhesh Bhandarib, Sangeeta Prakashb, Sylvester Mantihalb, Min Zhanga,c*
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aState
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Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, Jiangsu,
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China
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School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia
cInternational
Joint Laboratory on Food Safety, Jiangnan University, 214122 Wuxi, Jiangsu, China
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*Corresponding author: Dr. Min Zhang, Professor of School of Food Science and Technology, Jiangnan
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University, 214122 Wuxi, P. R. China.
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Tel.: 0086-510-85877225; Fax: 0086-510-85877225;
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E-mail: min@jiangnan.edu.cn
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Abstract: 3D food printing is an emerging technology with a potential to influence the food
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manufacturing sector. Rheological properties of food inks are critical for their successful 3D printing.
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However, the relationships between rheological properties and 3D printability have not been clearly
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defined in food systems. In this work, a gel model system composed of carrageenan-xanthan-starch was
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prepared for an extrusion-based 3D food printer. The 3D printing process was divided into three stages
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and the corresponding rheological properties of inks for each stage were determined, namely extrusion
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stage (yield stress, viscosity and shear-thinning behaviour), recovery stage (shear recovery and
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temperature recovery properties) and self-supporting stage (complex modulus G* and yield stress at room
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temperature). Finally, 3D printability of the model inks was systematically studied starting with printing
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lines/pentagram (one dimensional, 1D structure) to printing lattice scaffold (two dimensional, 2D
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structure) and finally printing cylinders (three dimensional, 3D structure). Results demonstrated that
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addition of starch and xanthan gum in k-carrageenan based inks increased inks’ gelation temperature
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(Tgelation), viscosity (within shear rate of 0.01 ~ 100 1/s), yield stress, G*, enhanced shear-thinning
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(thixotropic) behaviour and reduced time-dependence of modulus (temperature recovery). Rheological
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responses of yield stress (cross-over point where G (elastic modulus) equals to G (viscous modulus) in
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the stress sweep tests) and shear-thinning behaviour (viscosity decreased when shear rate increased) were
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closely related to ink’s extrudability. Inks’ gelation temperature (Tgelation) and time-dependent
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behaviour (gelation time, tgel) significantly affected their printability and shape retention performance.
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The mechanical strength of the ink is important to be self-supporting, especially for 3D structures.
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Insights achieved from this study could provide guidance on improving 3D printability of foods that use
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hydrocolloids as a printing aid.
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Keywords: 3D printing, rheological properties, gelation temperature, printability
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1. Introduction
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3D printing enable the fabrication of structures in a layer-by-layer pattern that are created in pre-
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designed files (Rayna & Striukova, 2016). Several advantages of 3D food printing have been reported,
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such as, customization of food structures, alteration of food texture, personalization of nutrition and use of
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various food sources (Liu, Zhang, Bhandari, & Wang, 2017; Sun, Zhou, Huang, Fuh, & Hong, 2015).
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Currently, there are four printing techniques that have been applied in food sector, namely extrusion
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based printing, selective sintering printing, binder jetting and inkjet printing (Liu et al., 2017). Extrusion
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based printing is so far the most frequently used for food printing as it is able to utilize a wide range of
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available food inks. It has been able to fabricate structures from slurry or mashed food materials like
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mashed potatoes (Liu, Bhandari, Prakash, & Zhang, 2018; Liu, Zhang, Bhandari, & Yang, 2018), dough
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(Severini, Derossi, & Azzollini, 2016), chocolate (Mantihal, Prakash, Godoi, & Bhandari, 2017), gel
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system (Cohen et al., 2009; Wang, Zhang, Bhandari, & Yang, 2017), and a combination of mashed
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potatoes and juice gel system (Liu, Zhang, & Yang, 2018). During fabrication process, the food inks are
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dispensed from the material reservoir by pneumatic, piston or screw driven system (Hamilton, Alici, & in
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het Panhuis, 2018; Lille, Nurmela, Nordlund, Metsä-Kortelainen, & Sozer, 2018; Yang, Zhang, Bhandari,
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& Liu, 2018). To improve printability in extrusion based printing, a good understanding of material’s
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rheological properties is required. The food ink must not only be easily extruded out through a narrow
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nozzle tip, but also possess enough mechanical strength to minimize deformation once being printed. Two
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strategies have been proposed to achieve the desired rheological behaviour. The first strategy is to prepare
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the inks with a low viscosity and yield stress that can be easily extruded, rapidly set through gelation
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process once extruded from the nozzle tip and possess a high mechanical strength that resists deformation.
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The second strategy is to prepare the inks with desired rheological properties, like shear-thinning
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behaviour which is desirable during extrusion and possess sufficient mechanical strength to withstand
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deposited structure (M’Barki, Bocquet, & Stevenson, 2017). In bio-printing, it has been reported that
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shear-thinning and thermo-reversible behaviour of inks are highly desirable. The shear-thinning behaviour
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enables the easy extrusion of inks. While the thermo-reversible behaviour enables the inks to quickly
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achieve enough yield strength to be capable of self-supporting through gelation (Wilson, Cross, Peak, &
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Gaharwar, 2017; Zhang et al., 2015). Usually, these rheological behaviour are often closely related with
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each other and cannot be varied separately. Thus a deep understanding of rheological properties of inks is
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important to enable a successful printing. Some researchers have investigated the effect of rheological
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properties on 3D printing behaviour. They highlighted the importance of the rheological behaviour of inks
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on 3D printability, like shear-thinning character, viscosity and yield stress (Li, Liu, & Lin, 2016; Liu et
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al., 2018; Sweeney, Campbell, Hanson, Pantoya, & Christopher, 2017). For some materials with thermo-
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responsive behaviour, like thermo-reversible hydrogels of k-carrageenan and chocolate, the
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gelation/solidify temperature is critical to determine appropriate printing temperature (Chung et al., 2013;
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Holzl et al., 2016; Mantihal et al., 2017; Suntornnond, An, & Chua, 2017). Although many researchers
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have emphasized the importance of rheological properties on 3D printing behaviour, few researchers have
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correlated them during the whole printing process, starting from extrusion to the final self-supporting
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stage.
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As 3D food printing is a very recent development, investigations focused on the fabrication of edible
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gels by 3D food printing technique are very limited (Cohen et al., 2009; Valérie Vancauwenberghe et al.,
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2017; Wang et al., 2017). Insights achieved from investigations involving 3D printing of edible gels could
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deepen our understanding about many 3D food printing processes which use hydrocolloids as additives to
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improve printing performance. Carrageenan and xanthan gum are widely used in the food industry,
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especially in starch-based food products (Chaudemanche & Budtova, 2008; Fakharian et al., 2015;
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Gladkowska-Balewicz, Norton, & Hamilton, 2014; Lin, Liang, & Chang, 2016; Liu et al., 2018; Mandala
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& Bayas, 2004). The k-carrageenan is a linear sulfated polysaccharide derived from red algae with
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alternating 3,6-anhydro-D-galactose and β-D-galactose-4-sulphate repetitive units (Tecante & Santiago,
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2012). This polymer illustrates rapid thermo-reversible behaviour and can form a firm and brittle gel
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structure. It can be easily dissolved in water when heated, presenting a random coil structure. During
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cooling process, it undergoes a conformational transition from coils to double helices followed by the
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aggregation of helices. This results in the formation of a brittle gel structure stabilized by hydrogen
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bonding between galactose (Liu, Chan, & Li, 2015; Wilson et al., 2017). Xanthan gum is an extracellular
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polysaccharide secreted by the micro-organism Xanthomonas campestris. Xanthan gum solutions are
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highly pseudoplastic with a strong shear-thinning and recovery behaviour. Viscosity rapidly decreases
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when shear stress increases, but after removal of shear force, the initial viscosity is recovered almost
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immediately. This property related to the formation of intermolecular aggregates by xanthan molecules is
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through hydrogen bonding in solution. The highly ordered network of entangled and stiff molecules
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presents a high viscosity at low shear stress, while it is progressively disrupted when shear stress is
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applied (Graham, 2009; Mandala et al., 2004; Morrison, Clark, Talashek, & Yuan, 2004). The strong
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shear-thinning and rapid recovery behaviour are highly desirable in extrusion based 3D printing as shear-
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thinning behaviour enables the easy extrusion of inks through a narrow opening and the rapid recovery
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behaviour allows the inks to quickly achieve enough mechanical strength after printing to resist
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deformation.
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This work was aimed at improving our understanding of correlation between rheological properties and
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3D printability and provide some information on 3D food printing of carrageenan-xanthan-starch
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multicomponent system. In this study, we firstly investigated the thermo-responsive behaviour of inks,
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following which several formulations were selected for determination of rheological properties and
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evaluation of 3D printability. The 3D printing process was divided into three stages, namely extrusion
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stage, recovery stage and self-supporting stage, and corresponding rheological properties of inks for each
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stage were determined. Finally, 3D printability of model ink formulations was evaluated based on the
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ability to print 1D structure (line, pentagram), 2D structure (lattice scaffold) and 3D structure (cylinder).
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2. Materials and methods
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2.1 Materials
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Food grade xanthan gum (Batch: 345216) and kappa-carrageenan (k-carrageenan, Batch: 336115) were
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purchased from Melbourne Food Ingredient Depot Co. Ltd, Brunswick, Australia. Xanthan gum and k-
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carrageenan contain no fillers, preservatives, bulking agents and flavors. Potato starch with moisture
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content of 16.52±0.23 g/100g (w.b.) was provided by Simplot Co. Ltd, Devonport, Australia.
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2.2 Inks preparation
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Ink formulations were prepared by following the procedure as shown in Fig. 1A. Required amount of
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k-carrageenan, xanthan gum, and potato starch were firstly well mixed and dissolved in water whilst
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constant stirring. The mixture (100 mL) were stirred for 30 min at 1200 r/s using a stirrer (IKA@ RW 20,
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John Morris Scientific Pty Ltd, Australia). During this process, 0.1% food color was incorporated to the
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mixture system. Afterwards, the mixtures were incubated for 30 min at 90°C using a water bath and were
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homogenized for 4 min at 3000 r/s (5702 R, Eppendorf Co. Ltd, Germany) to remove the air bubbles
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introduced during the stirring process. For the formulation containing only potato starch, continuous
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stirring was conducted when heating to avoid syneresis/separation of water. The prepared inks were then
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stored in a beaker in a refrigerator at 4 °C and evaluated within two days.
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A 23 factorial design was used to prepare the ink formulations. Tab. 1 describes the concentration levels (-1,
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+1) for k-carrageenan, xanthan gum, and potato starch. The experimental matrix including eight
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formulations in total can be seen in Tab. 2. In addition, as a comparative study, inks containing only 0.25%
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xanthan gum, 1% k-carrageenan, and 2% potato starch were also prepared.
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2.3 Rheological analysis
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The rheological behaviour of ink formulations were characterized using an AR-1500 rheometer (TA
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Instruments Ltd, UK) with a plate–plate geometry (40 mm diameter, 0.2 mm gap) and a very thin layer of
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silicon oil in the trap to prevent the moisture evaporation. The formulations were firstly heated up to 60°C
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before testing to form a flowable state. A plastic dropper was used to load samples with same number of
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drops. Afterwards, the samples were equilibrated at initial measurement temperature for 7 min to reach a
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steady state, unless otherwise stated. The shear-viscosity tests were conducted in flow ramp mode with
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the shear rate increasing from 0.01 to 100 1/s at temperature of 35°C, 40°C and 45°C. The yield stress
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measurements were performed in oscillation stress sweep mode at 1 Hz from 1 to 1000 Pa (at testing
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temperature of 35°C, 40°C and 45°C) or to the upper limit of stress achievable for the rheometer (at
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25°C). The yield stress of inks was determined as the cross-over point where elastic modulus (G) equal to
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viscous modulus (G) (Wilson et al., 2017). Temperature ramps were performed at 10 s-1 from 65°C to
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30°C with a cooling rate of 3°C/min. Gelation temperature (Tgelation) was determined by extrapolating
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the high and low temperature asymptotes of the viscosity and specifying the temperature at which these
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intersects (Barrera, Florián-Algarin, Acevedo, & Rinaldi, 2010). Rotational recovery tests were applied to
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characterize the shear recoverability of inks at 40°C by using a low shear rate of 1 1/s for 180 s, followed
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by a high shear rate at 100 1/s for 120 s and finally at a low shear rate of 1 1/s for 180 s. Shear
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recoverability of inks was determined as the percentage of viscosity obtained during the first 30s in the
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third step after high shear rate (100 1/s) based on the average viscosity obtained in the first step
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(Achayuthakan & Suphantharika, 2008). Temperature recovery tests (coined by the authors) were
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conducted to characterize the time dependence of complex modulus (G*, G*=( G2+ G2)0.5) when
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temperature changed from printing temperature (35°C, 40°C, 45°C and 50°C) to room temperature
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(25°C). Frequency sweep analysis was performed at 25°C with angular frequency ranging from 1 to 100
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rad/s at a constant deformation of 0.1% strain (within the linear viscoelastic range, LVR). Three replicates
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were conducted and the average data were used to plot the curves.
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2.4 3D printing process
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A syringe type extrusion-based 3D printer (Choc Creator 2.0 Plus, Choc Edge Co. Ltd, UK) was used
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to assess the printability of the inks. The desktop printer comes with a syringe-based deposition unit. The
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syringes is inserted into one material barrel. A heating unit is used to control the printing temperature. The
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printer could be controlled by a computer or the graphical user interface unit. A photograph of the printer
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was given in Fig. 1B.
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Room temperature of 25°C was applied in the printing process. The printing temperature was set at
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35°C, 40°C, 45°C and 50°C through the temperature-controlled nozzle and the platform was held at room
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temperature (25°C). The inks were firstly heated up to 60°C to form a flowable state and were then
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poured into a metal syringe. During this process, it was made sure that no air bubble was included. The
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syringes filled with inks were then incubated at printing temperature (35°C, 40°C, 45°C and 50°C) for at
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least 1 hour prior to be used for deposition process. Printability tests, that is 1D printing (line, one-layer
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pentagram), 2D printing (three-layer of lattice scaffold) and 3D printing (hollow cylinder) were printed at
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the same condition (nozzle diameter 0.8 mm, layer height 0.8 mm, federate and printing rate 22 mm/s,
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and solid infill). The stereolithography (stl) models (line, pentagram, lattice scaffold, and hollow cylinder)
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were designed with Rhinoceros 5.0 (educational version, Robert McNeel & Associates, Seattle, US) and
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detailed information are shown in Fig. 1C and Fig. 1D.
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2.5 Printability evaluation
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The printability of inks under various printing temperature was evaluated by consistency of lines,
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average area of interconnected channels in lattice scaffold, and wall thickness of hollow cylinder. Images
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of the printed constructs were captured once the fabrication process was completed using a 16 megapixel
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(16 MP (f/2.0, 1/2.8", 1.12µm) camera (Nubia Z11, Nubia Technology Co., Ltd., Shenzhen, China) with a
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ruler beside the construct. The average area of interconnected channels within lattice scaffolds and wall
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thickness of hollow cylinder were analyzed using the ImageJ software. The images were firstly set a scale
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and were then converted to 8 bit. After adjusting the threshold, the area of interconnected channels of
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lattice scaffold could be automatically measured and analyzed. The “Straight Line” tool was used to
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calculate the wall thickness of hollow cylinder.
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2.6 Statistical analysis
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The statistical software SPSS was used to analyze data and Origin 9.0 was used to plot the graphs.
Differences of p < 0.05 were considered to be significant with the application of Duncan’s test.
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3 Results and discussion
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3.1 Choice of materials and their properties
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In extrusion-based 3D printing, inks must easily flow through a nozzle tip, and should also possess
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enough mechanical strength to resist deformation after deposition. For the inks to have desired rheological
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properties, previous researchers have prepared inks with sufficient yield strength that enable the printed
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constructs capable of self-supporting, such as mashed potatoes (Liu et al., 2018), baking dough (Yang,
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Zhang, Prakash, & Liu, 2018) and orange juice gel (Roknul, Zhang, Mujumdar & Yang, 2018). However,
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the inks with high mechanical strength always characterized by high viscosity and yield stress, which
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could easily block the nozzle tip and result in the failure of printing process. To address this problem, inks
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with a strong shear-thinning and a rapid shear recovery properties are required (Paxton et al., 2017).
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Another desirable property of ink is thermo-responsive behaviour, which means that ink’s viscosity is
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reduced considerably at a high temperature but rapidly develops significant mechanical strength to resist
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deformation once being extruded out from a nozzle due to the temperature difference between nozzle
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cavity and fabrication platform. This method is frequently used in chocolate printing (Mantihal et al.,
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2017) and thermo-reversible gels printing (Ouyang, Yao, Zhao, & Sun, 2016; Wilson et al., 2017). In our
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study, the two strategies of strong shear-thinning behaviour/rapid shear recovery and thermo-responsive
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behaviour were adopted to fabricate the multicomponent gel system comprised of k-carrageenan, xanthan
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gum and starch.
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k-carrageenan and xanthan gum are both widely used in the food industry, especially in starch-based
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food products (Chaudemanche et al., 2008; Fakharian et al., 2015; Gladkowska-Balewicz et al., 2014; Lin
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et al., 2016; Mandala et al., 2004). As previously mentioned in the introduction k-carrageenan has a rapid
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thermo-reversible behaviour while xanthan gum is highly pseudoplastic with a strong thixotropic and
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recovery behaviour. Hence, it would be interesting to observe how these two ingredients with the desired
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characteristics required for good printability, perform in a multicomponent gel system of k-carrageenan-
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xanthan-starch.
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Because the ink illustrates a thermo-reversible characteristic, it is important to determine its gelation
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temperature (Tgelation) to set a proper printing temperature in the nozzle cavity. Therefore, in our work,
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Tgelation of inks were firstly determined to select suitable ink formulations for further rheological tests
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and 3D printability evaluation as the upper limit temperature of nozzle cavity was 50°C. Subsequently,
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the rheological properties of the selected inks were correlated with their 3D printability. We divided the
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extrusion-based printing process into three stages, and rheological properties of inks corresponding to
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each stage were evaluated (Fig. 1A).
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The first stage of printing is an extrusion stage. The inks’ viscosity, yield stress and thixotropic (shear-
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thinning) behaviour were characterized to evaluate their extrudability during the extrusion process. The
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second stage is the recovery stage. During this stage, the inks were exposed to high shear rates and
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experienced temperature variations when they were extruded out from the nozzle cavity (printing
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temperature: 35°C, 40°C, 45°C and 50°C) to the deposited platform at room temperature (25°C). Here, we
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define the shear recovery to mimic the recoverability of inks after being experienced the high shear rates
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and the temperature recovery to characterize the time dependence of viscoelastic behaviour of inks during
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temperature variations. Therefore, in a rheological test mimicked shear recovery tests applying an
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alteration of high and low shear rates to the inks and temperature recovery tests applying an alteration of
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printing temperature and room temperature were conducted in this stage. The third is self-supporting
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stage. After finishing the deposition process, inks would be exposed at room temperature. During this
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stage, the yield stress and G* are both important in determining the self-supporting behaviour of
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deposited parts. Finally, we evaluated the printability of inks based on 1D structure (line, pentagram), 2D
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structure (lattice scaffold) and 3D structure (cylinder), and correlated the printability behaviour of inks
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with their rheological properties (Fig. 1A).
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3.2 Thermo-reversible characteristics of inks
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The thermo-responsive behaviour of inks with different compositions were evaluated by rheological
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tests. As shown in Fig. 2A, viscosity of 1% k-carrageenan solution (kC(1)) increased rapidly at
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temperature below
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transition from coils to double helices followed by the aggregation of helices (Liu, et al., 2015; Parker,
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Brigand, Miniou, Trespoey, & Vallée, 1993; Shchipunov, 2003). The cross-linking in k-carrageenan gel is
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associated with the formation of double helices and their aggregates. This process is partially governed by
35°C due to the gelation of k-carrageenan during cooling involving a conformational
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micro crystallization, and the double helices constraining further aggregation and propagation during
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gelation evolution (Liu et al., 2015). Comparatively, the viscosity of the 2% starch solution (S(2)) and
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0.25% xanthan solution (X(0.25)) changed little when temperature decreased, indicating that they were
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not responsible for the thermo-responsive behaviour of multicomponent inks. In addition, the inks
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containing k-carrageenan (kC(1)X(0.25) and kC(1)X(0.25)S(2)) represented similar viscosity change
250
profiles as kC(1), indicating that k-carrageenan played a more important role in the thermo-responsive
251
behaviour of the multicomponent inks.
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As shown in Fig. 2B, the gelation temperature (Tgelation) was recorded from the viscosity
253
measurements from temperature ramp-down curve by extrapolating the high and low temperature
254
asymptotes of the viscosity and specifying the temperature at which these intersect (Barrera et al., 2010).
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As expected, Tgelation increased significantly when k-carrageenan concentration increased (Tab. 3). The
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Tgelation of inks containing 2% k-carrageenan occurred above 44°C, while the inks containing 1% k-
257
carrageenan showed a gelation point below 40°C. This was because an increased k-carrageenan
258
concentration would increase the amount of available interaction sites for hydrogen bonding between k-
259
carrageenan polymer chains and thus making it easier for the formation of double helices and their
260
subsequent aggregation (Fakharian et al., 2015; Gladkowska-Balewicz et al., 2014; Wilson et al., 2017).
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During extrusion tests it was found that inks containing 2% k-carrageenan easily gelled due to the high
262
Tgelation and high viscosity, causing the nozzle tip to block. Therefore, only the inks containing 1% k-
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carrageenan were selected for further evaluation of rheological properties and printability tests.
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As seen from Fig. 2A and Tab. 3, starch addition led to the increase of viscosity and Tgelation. This
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might be related to the excluded volume effect of starch during which the presence of swollen starch
266
molecules leads to the reduction of accessible water molecules which are held by k-carrageenan. This
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phenomenon results in the formation of some zones with elevated k-carrageenan concentration,
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facilitating the increase of viscosity, elastic modulus and the rates of ordering and aggregation
269
(Chaudemanche et al., 2008; Mandala et al., 2004). The incompatibility between polymers when one of
270
the ingredients acts as gelling agent could result in the dramatic change in gel properties, including an
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increased Tgelation and modulus (Fakharian et al., 2015; Lorenzo, Zaritzky, & Califano, 2015).
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Addition of xanthan gum also increased the Tgelation and viscosity of inks (Fig. 2A and Tab. 3). This
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was because the conformational transition of xanthan molecules when temperature decreased from a more
274
flexible and disordered structure to a rigid ordered state which can form intermolecular associations and
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network structure of weakly bound molecules (Graham, 2009; Morris, Rees, Young, Walkinshaw, &
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Darke, 1977; Morrison et al., 2004).
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3.3 Rheological properties of inks governing extrusion stage
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3.3.1 Flow initiation analysis determined by yield stress at printing temperature
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The extrusion process in 3D printing is easier for ink with a low yield stress (Sweeney et al., 2017).
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Yield stress also reflects the mechanical strength of inks as it supports subsequent stacked layers during
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post-printing condition (Liu et al., 2018). In our study, as the inks went through a rapid temperature
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change from printing temperature in nozzle cavity (35°C, 40°C, 45°C, and 50°C) to room temperature at
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deposited platform (25°C), the flow stresses were determined at printing temperature and room
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temperature, respectively.
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Yield stress (flow stress) is critical to ink’s extrudability as it closely relates to the minimum force
287
required to initiate a flow of ink (Li et al., 2016; Paxton et al., 2017). The minimum pressure required to
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start a flow for the ink with a yield stress can be described by
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Sweeney et al., 2017). Where Pmin means the minimum pressure required, L means the nozzle length, D
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means the nozzle diameter, and τyield means the yield stress of inks. During extrusion processes, a low
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(Pospischil et al., 2014;
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yield stress is highly desirable as the extrusion is not continuous but starts and stops frequently during
292
printing. A high yield stress requires the printer to produce a larger force to exceed the yield stress. This
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necessities a more robust extrusion system to be capable of constantly generating such high pressure in a
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controlled way. (Lewis, 2006; Lewis, Smay, Stuecker, & Cesarano, 2006).
295
Yield stresses of inks was determined as the cross-over point where G equals to G (Fig. 2C) (Wilson
296
et al., 2017). The yield stress of inks at different printing temperature are illustrated in Tab. 4. As
297
expected, yield stresses for inks were decreased when temperature increased. In particular, for the inks of
298
kC(1)X(0.5)S(2) and kC(1)X(0.25)S(2), yield stresses decreased sharply from 551.3 Pa to 36.9 Pa and
299
from 134.2 Pa to12.0 Pa when temperature increased from 35°C to 45°C, indicating that a higher
300
extrusion force is required to initiate the flow of inks at a low temperature. The thermo-responsive
301
behaviour of k-carrageenan contributed the decrease of yield strength. Tgelation of inks of
302
kC(1)X(0.5)S(2) and kC(1)X(0.25)S(2) were 38.2°C and 36.9°C (Tab. 3), respectively. The k-
303
carrageenan molecules dissolve in water and present a random coil structure when temperature is above
304
Tgelation, while, at below Tgelation, they change to double helices followed by the aggregation of helices
305
finally forming a gel (Tecante et al., 2012; Wilson et al., 2017).
306
The increased amount of xanthan gum increased the yield stress of inks. For example, at 35°C, the
307
yield stress was 39.2 Pa for kC(1)X(0.25)S(0) and 80.2 Pa for kC(1)X(0.5)S(0), respectively. Addition of
308
2% starch also increased the yield stress from 80.2 Pa for kC(1)X(0.5)S(0) to 551.4 Pa for
309
kC(1)X(0.5)S(2). This increase might be attributed to the excluded volume effect of starch, where the
310
existence of starch molecules in the mixture results in a decrease of the accessible water molecules that
311
can be captured by k-carrageenan. Thus, a relative increased k-carrageenan concentration enhanced its
312
aggregation rates resulting in increased yield stress and viscosity (Chaudemanche et al., 2008;
313
Gladkowska-Balewicz et al., 2014; Lin et al., 2016).
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315
3.3.2 Shear-thinning behaviour
316
After overcoming the yield stress, the required pressure to maintain a continuous flow of inks would
317
depend on their viscosity and shear-thinning behaviour. One model that frequently used for shear-thinning
318
fluids is the power-law (
319
Where ƞ means viscosity, K means the consistency index, γ̇ means shear rate, and n means flow index that
320
describes the material’s properties of shear thinning, thickening or remain Newtonian. For n < 1 fluids
321
shear thin and a smaller n indicates a more highly shear-thinning behaviour (Paxton et al., 2017; Wilson et
322
al., 2017). Ink’s shear-thinning behaviour could be quantified by fitting the viscosity profiles (Fig. 3) to
323
Power Law equation. The coefficients K and n (Tab. 4), could help us to better understand the ink’s flow
324
behaviour according to the equation
325
tube, P means the pressure drop, and l means the length of tube length. A low K and n are advantageous
326
to require a low pressure for a continuous flow (Lewis, 2006; Lewis et al., 2006; Liu et al., 2018). The
327
shear viscosity and stress profiles of inks at different printing temperature are illustrated in Fig. 3. All inks
328
present shear-thinning behaviour, indicated by a reduction in viscosity and an increase in shear stress
329
when shear rate was increased. For all inks, K decreased over an increasing temperature. Specifically, for
330
the ink of kC(1)X(0.5)S(0), the K were 6.19 Pa·sn, 3.14 Pa·sn, and 2.68 Pa·sn at temperature of 35°C,
331
40°C, and 45°C, respectively. Again, this was related to the thermo-reversible behaviour of k-carrageenan
332
(Wilson et al., 2017; Yang, Yang, & Yang, 2018).
) (Rezende, Bartolo, Mendes, & Filho, 2009; Sweeney et al., 2017).
. Where Q means the flow rate, R means the radius
333
Addition of xanthan gum increased K at all tested temperature (Tab. 4). Added 2% potato starch also
334
increased ink’s viscosity (Fig. 3) and K (Tab. 4). Again, this might be due to the excluded volume effect
335
of starch in the system as discussed above (Chaudemanche et al., 2008; Gladkowska-Balewicz et al.,
336
2014; Lin et al., 2016).
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As seen from Tab. 4, the flow index (n) was less than one, suggesting that inks exhibits shear thinning
338
behaviour. The n presents no regular change with temperature variations. However, addition of xanthan
339
gum and potato starch obviously decreased the values of n, meaning that shear thinning was more
340
pronounced. Specifically, at 35°C and 45°C, n decreased from 0.58 and 0.71 for kC(1)X(0.25)S(0) to 0.50
341
and 0.61 for kC(1)X(0.5)S(0) due to the increased amount of xanthan gum, and from 0.58 and 0.71 for
342
kC(1)X(0.25)S(0) to 0.47 and 0.48 for kC(1)X(0.25)S(2) due to the addition of starch. As reported, the
343
xanthan gum solutions present highly pseudoplastic behaviour. An increase of shear rate will
344
progressively reduce the viscosity, but upon the removal of shear stress the viscosity will recover rapidly.
345
This was because xanthan molecules can form intermolecular aggregates through hydrogen bonding and
346
polymer entanglements that presents high viscosity at low shear rates, and the structure are disrupted
347
rapidly with the application of high shear rates (Graham, 2009; Morrison et al., 2004). A more
348
pronounced shear thinning effect was also reported after addition of starch in k-carrageenan system
349
(Fakharian et al., 2015).
350
351
3.4 Rheological properties of inks governing recovery stage
352
3.4.1 Shear recovery behaviour
353
During printing, the inks experience the shear force and temperature changes when being extruded out
354
from the nozzle tip. Thus, we divided the ink’s recovery properties into shear recovery and temperature
355
recovery.
356
Here, as illustration, the shear recovery tests were conducted at a constant temperature of 40°C to
357
investigate the effect of individual shear forces on the viscosity changes. Thixotropic inks are highly
358
desirable for extrusion printing. In these inks, the viscosity is reduced rapidly with the application of shear
359
force but recovers quickly after the removal of shear force (Lille, Nurmela, Nordlund, Metsä-Kortelainen,
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& Sozer, 2017). To mimic the shear conditions during extrusion printing, the inks were firstly subjected to
361
a low shear rate of 1 1/s to simulate the low extrusion rate of initial extrusion state in the syringe barrel
362
part. Subsequently, the shear rate was increased to 100 1/s to simulate the shear force generated in the
363
nozzle tip part during extrusion. Finally, the shear rate was again decreased to 1 1/s to test the recovery
364
behaviour of inks to simulate post-printing condition because the rheometer cannot test viscosity at zero-
365
shear. Fig. 4A illustrates the shear recovery behaviour of viscosity of the inks under an alterations of low
366
and high shear rates. All inks’ viscosity decreased significantly at high shear rate and recovered rapidly at
367
low shear rate, and quickly reached a stabilized state within 30s. In the case of kC(1)X(0.5)S(0), viscosity
368
dramatically decreased from 4.33 Pa.s at 1 1/s to 0.36 Pa.s at 100 1/s, and then rapidly built up to 3.92
369
Pa.s in 10s (90.53% recovery of the initial viscosity). If it was allowed 30s, a 98.91% recovery was
370
obtained. For the ink kC(1)X(0.5)S(2), the recovery percentage was 75.56% in 10s and was 83.09% in
371
30s, but it could not increase further even it was given a longer time. This rapid and reversible viscosity
372
response make the inks suitable for 3D printing as they could be easily extruded out while rapidly
373
recovered enough mechanical strength necessary to support the next extruded layer. Fig. 4B shows the
374
recovery percentage of inks within 30s after removal the high shear rate (100 s-1). After addition of starch,
375
the recovery decreased from 91.30% for kC(1)X(0.25)S(0) to 78.50% for kC(1)X(0.25)S(2), from 98.91%
376
for kC(1)X(0.5)S(0) to 83.09% for kC(1)X(0.5)S(2), respectively. This might be related to the excluded
377
volume effect of starch in the multicomponent gel system (Chaudemanche et al., 2008; Mandala et al.,
378
2004). The incompatibility between polymers when one of the ingredients acts as gelling agent could
379
result in the dramatic change in recoverability (Fakharian et al., 2015; Lorenzo et al., 2015).
380
Advantageously, with the increased amount of xanthan gum, the recovery percentage increased form
381
91.30% for kC(1)X(0.25)S(0) to 98.91% for kC(1)X(0.5)S(0), from 78.50% for kC(1)X(0.25)S(2) to
382
83.09% for kC(1)X(0.5)S(2), respectively. The increase of cross-linking density by physical bonds
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because of the increased addition of xanthan gum might contribute to the reversible-structure behaviour.
384
In addition, this was also probably because the formation of intermolecular aggregates of xanthan
385
molecules stabilized by hydrogen bonding presents a high viscosity at low shear rates, and the structures
386
are disrupted rapidly with the application of high shear rates, but upon the removal of shear stress the
387
viscosity recovered rapidly (Graham, 2009; Morrison et al., 2004).
388
3.4.2 Temperature recovery behaviour
389
During 3D printing process, the inks experienced a rapid temperature change from printing temperature
390
(nozzle cavity temperature of 35°C, 40°C, 45°C, and 50°C) to the room temperature (25°C). The gelation
391
kinetics of inks (temperature recovery) were investigated through a temperature cycle test (Fig. 5). To
392
simulate the temperature changes experienced by the inks during 3D printing process, the complex
393
modulus (G*, it provides an overall deformation resistance (Huang, 2018)) of inks were firstly recorded at
394
printing temperature (35°C, 40°C, 45°C, and 50°C) for 120s, and the gelation behaviour of inks were then
395
recorded at room temperature (25°C) for 300s,. Generally, the inks responded rapidly to the decrease of
396
temperature indicated by the sharp increase of G*, but it took a certain time for the inks to reach a plateau
397
in G*. The time necessary to reach the G* plateau was defined as tgel. It was determined as the crossover
398
point of two tangents respectively fitted to the plateau-region and G*-increase regions of the curves at
399
room temperature (25°C), as shown in Fig. 5A. We emphasize that the length of tgel is critical to the
400
shape retention of extruded filaments and final printed parts. A long tgel would definitely cause the
401
spreading deformation of filaments and printed part as inks cannot achieve a sufficient mechanical
402
strength within a short time to be capable of self-supporting. The tgel of inks at different temperature is
403
shown in Fig. 5F. As expected, for all inks the tgel increased with an increasing temperature. In
404
particular, the tgel increased from 77s at 35°C to 98s at 50°C for the ink of kC(1)X(0.25)S(2). This was
405
obvious because biopolymer molecules require a long time to form a gel like structure after being
18
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subjected to a high temperature. Note that the tgel decreased with the addition of starch, for example at
407
45°C, the tgel decreased from 106s for kC(1)X(0.25)S(0) to 91s for kC(1)X(0.25)S(2) and from 96s for
408
kC(1)X(0.5)S(0) to 85s for kC(1)X(0.5)S(2), respectively. This is probably because starch molecules are
409
not thermo-responsive and their excluded volume effect lead to a reduction of the accessible water for k-
410
carrageenan molecules. This resulted in the formation of a relative partial high concentration of k-
411
carrageenan making it easier to form a gel (Chaudemanche et al., 2008; Gladkowska-Balewicz et al.,
412
2014; Lin et al., 2016). In addition, the interfacial phenomena between starch granules (solid phase) and
413
the continuous phase medium might be also accounted for this fact, as the interfacial surface-tension is
414
greater at lower temperature which can produce a rigid interface at the boundary solid-liquid phases (Shi
415
& BeMiller, 2002). With the increase of xanthan gum, the tgel also decreased. This may be related to the
416
conformational transition of xanthan molecules from a more flexible and disordered state to a rigid rod
417
state of network structure (Graham, 2009; Morrison et al., 2004).
418
419
3.5 Rheological properties of inks governing self-supporting stage
420
To be capable of self-supporting after deposition, the complex modulus (G*) and yield stress are
421
important (Huang, 2018; Liu et al., 2018; Wilson et al., 2017). G* means solid-like property reflecting the
422
ability to resist compression deformation and the mechanical strength (Huang, 2018). Apparently, the inks
423
with sufficient mechanical strength and yield stress illustrate excellent self-supporting behaviour (Lille, et
424
al., 2017; Yang et al., 2018). As seen from Fig. 6, G′ was obviously higher than viscous modulus (G″) and
425
they both exhibited frequency-dependent behaviour. The values of G′, G″ and G* at 10 rad/s for inks are
426
presented in Tab. 3. Increased amount of k-carrageenan significantly increased the G* from 64.6 Pa for
427
kC(1)X(0.25)S(0) to 694.3 Pa for kC(2)X(0.25)S(0), while decreased the tan (tan= G″/ G′) from 0.77 to
428
0.20, respectively. Similar trend for all inks were observed. This was expected since at a high
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concentration k-carrageenan is easier to transform into a denser gel state due to the formation of a network
430
structure induced by the aggregation of double helices (Liu et al., 2015). Addition of potato starch also led
431
to the significant increase of G* for all inks. Again, this might be because the excluded volume effect of
432
starch in the system decreased the amount of accessible water molecules to k-carrageenan thus increasing
433
its partial concentration (Chaudemanche et al., 2008; Gladkowska-Balewicz et al., 2014; Lin et al., 2016).
434
The G* for the ink of kC(1)X(0.25)S(2) rapidly increased from 590.1 Pa to 836.6 Pa when xanthan gum
435
increased by 0.25 g (kC (1)X(0.5)S(2)), while their solid-like character was similar with tan value of
436
0.21 for kC(1)X(0.25)S(2) and 0.23 for kC(1)X(0.5)S(2). This means that the increase in the amount of
437
xanthan in presence of starch produced more rigid networks maintaining similar degree of viscoelasticity.
438
However, the increased amount of xanthan in the inks without starch significantly increased G′, G″ and
439
G* but tan noticeable decreased forming a more compact network. Thus, the presence of starch does not
440
improve the energy stability of bonds in carbohydrate network, which could be related with interfacial
441
effects.
442
Another important parameter reflecting the resistance to deformation of inks is yield stress at self-
443
supporting stage (Lille, et al., 2018; Liu et al., 2018). During this stage, yield stress reflects the ink’s
444
performance to retain its shape under gravity and under the force given by upper material layers on top of
445
it. Many publications have reported that G′, G* and yield stress were critical to self-supporting ability and
446
maintain deposited information (Lille et al., 2018; Zhang et al., 2015; Huang, 2018). Here, please note
447
that we tested the yield stress of inks at printing temperature (35°C, 40°C, 45°C, and 50°C) and at room
448
temperature (25°C). We emphasize that the yield stress at printing temperature was used to describe the
449
extrusion behaviour during extrusion stage (section 3.3.1), while the yield stress at room temperature was
450
used to describe the self-supporting behaviour during post-printing. An oscillation stress sweep at 1 Hz
451
from 1 to the upper limit of stress achievable for the rheometer (at 25°C) was conducted. It was found that
20
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452
the yield stress for all inks was above 1000 Pa (data not shown), while the upper limit of stress of the
453
rheometer was around 2000 Pa. Therefore, it is possible that the yield stress obtained at such condition
454
might not be reliable thus it was not reported.
455
It is worth mentioning that the G′, G* and yield stress obtained at room temperature (25°C) might not
456
really reflect the deformation resistance performance as they were determined at a steady state at 25°C.
457
While, in our tests, inks went through a rapid temperature change from printing temperature (35°C, 40°C,
458
45°C, and 50°C) to room temperature (25°C). It’s not possible for the inks to reach a steady state at such a
459
short time (tgel, section 3.4.2). However, we emphasize that they can still give us useful information
460
about ink’s self-supporting behaviour for the deposited objects after reaching a steady state and in the
461
subsequent storage period.
462
3.6 Printability evaluation of inks
463
3.6.1 One-dimensional structure (1D structure)
464
To evaluate ink’s printability, a variety of constructs was printed. Fig. 7 shows some images of 1D
465
structures of lines and pentagram with different angles. As seen from Fig. 7A, some broken lines with a
466
very rough surface were captured when the ink of kC(1)X(0.5)S(2) was printed at 35°C. This was because
467
its high yield stress and K at 35°C resulting in its poor extrudability. At 35°C, the yield stress and K of
468
kC(1)X(0.5)S(2) were 551.3 Pa and 23.88, respectively (Tab. 4), significantly higher than those values at
469
other temperatures. As described in section 3.3.1, the yield stress is important to enable ink’s extrudability
470
from a narrow opening as it reflects the minimum pressure required to initiate the flow of inks, below
471
which the inks show more solid-like behaviour rather than liquid-like behaviour. Inks cannot be extruded
472
out unless the driving pressure larger than yield stress (Li et al., 2016; Paxton et al., 2017). A high K also
21
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473
showed an adverse effect to maintain the flow of inks as discussed in section 3.3.2 (Lewis, 2006; Lewis et
474
al., 2006).
475
Pentagrams with different angles were printed using ink of kC(1)X(0.25)S(2) (Fig. 7B). The overlap
476
problem was more serious in the fabrication of pentagram with a sharper angle (Fig. 7B(b)). As seen from
477
Fig. 7B(a), some areas were overlapped and amount of inks was doubled, which would cause the
478
accumulation of layer height errors during printing process, especially in the fabrication of 3D structures.
479
To address the angle printing deviations, two methods have been proposed: first one was to avoid the
480
sharp angle printing and the second one was to reduce the extrusion rate to half value at this overlapping
481
area or double the nozzle moving speed (He et al., 2016).
482
Pentagrams with a sharp angle printed at different temperature using ink of kC (1)X(0.25)S(2) are
483
shown in Fig. 7C. The overlap problem was more prominent for the samples fabricated at a high
484
temperature, especially at 50°C. This was because, during printing, the inks experienced a rapid
485
temperature change from a printing temperature in the nozzle cavity (35°C, 40°C, 45°C and 50°C) to the
486
room temperature (25°C), during which the inks were required to rapidly turn into a gel-like structure. As
487
illustrated in Fig. 5, the gelation of inks is a time-dependent process and a high temperature requires a
488
long tgel time. From rheological study, an increased tgel time was obtained from 77s at 35°C to 98s at
489
50°C (Fig. 5F). Generally, if the inks were still in an liquid like form after being extruded onto the
490
platform they would experience continuous spreading as they lack enough mechanical strength to be
491
capable of self-supporting.
492
493
3.6.2 Two-dimensional structure (2D structure)
494
Lattice scaffold is commonly used in bio-printing field and many researchers have used it to evaluate
495
the printing behaviour of bio-inks (He et al., 2016; Li et al., 2016; Ouyang et al., 2016; Paxton et al.,
22
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2017). Here, three layers of lattice scaffold (43 mm43 mm, crosshatch infill with 7 mm spacing between
497
fibers, 2.4 mm height, Fig. 1B) was printed. As seen from Fig. 8A and Fig. 8C, for all tested inks, the
498
area of interconnected channels decreased when printing temperature increased from 35°C to 50°C. This
499
was expected as inks being extruded out from the nozzle cavity at a higher temperature require a longer
500
gelation time tgel to achieve sufficient mechanical strength to self-support after being deposited onto
501
platform at room temperature (Fig. 5F). In particular, for the scaffolds printed object using
502
kC(1)X(0.25)S(0), the average area of interconnected channels decreased from 32.29 mm2 to 16.58 mm2
503
when temperature increased from 35°C to 50°C, even some interconnected channels were disappeared at
504
50°C. This was expected as tgel time increased from 86s at 35°C to 110s at 50°C for the ink of
505
kC(1)X(0.25)S(0). During the gelation process, the extruded filaments deformed continuously due to
506
insufficient yield strength to resist gravity and the force given by the subsequent layers. From
507
experiments, it was observed that inks showed three printing behaviour under different printing
508
temperature. If the ink is printed at a high temperature, it will present droplet morphological form at the
509
nozzle tip and the fabricated interconnected channels would show obvious chambers with a rounded
510
corner due to the fusion of adjacent layers, as the case of cylinders fabricated at 45°C and 50°C using ink
511
kC(1)X(0.25)S(0) and kC(1)X(0.5)S(0) (Fig. 8A and Fig. 8B). If the ink is deposited at a proper
512
temperature, the extruded filaments would be smooth and uniform, and an almost equal to square shapes
513
of interconnected channels would be created, like cylinders fabricated at 40°C using ink kC(1)X(0.25)S(2)
514
and kC(1)X(0.5)S(2) (Fig. 8A and Fig. 8B). In contrast, if the ink is fabricated at a low temperature, it
515
would result in irregular filaments forming fractured interconnected channels with a rough surface
516
structure. Sometimes it would even cause the congestion of nozzle tip, which was indicated by the
517
cylinder printed at 35°C using ink kC(1)X(0. 5)S(2) (Fig. 8A and Fig. 8B).
23
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518
Generally, it is recommended that the nozzle cavity temperature should be slightly higher than
519
Tgelation to ensure both the easy extrusion of inks and the rapid occurrence of gelation to form enough
520
mechanical strength to self-support once being extruded out from nozzle tip resulting from temperature
521
variation (Li, et al., 2016; Ouyang, et al., 2016). For instance, the scaffolds printed at 40°C using
522
kC(1)X(0.5)S(2) presented an smooth and uniform structure with an almost equal to square shapes of
523
interconnected channels. This was because the nozzle cavity temperature of 40°C was slightly higher than
524
the ink’s Tgelation (38.2°C), making it easier to gel rapidly once being extruded out form nozzle (tgel-
525
77s). In contrast, the cylinder printed at 35°C using kC(1)X(0.5)S(2) (Tgelation 38.2°C), presented a
526
rough surface structure and a larger interconnected channels (52.66 mm2) than desired (49.00 mm2) (Fig.
527
8A and Fig. 8C). Theoretically, when the ink of kC(1)X(0.25)S(0) was printed at 35°C, scaffolds with
528
regular square shapes should be obtained as the printing temperature was close to its Tgelation (35.4°C).
529
However, a smaller area of interconnected channels (32.29 mm2) than target (49 mm2) with a round
530
corner was still obtained (Fig. 8C). Several factors below might be accounted for this fact. Firstly, the
531
yield stress of kC(1)X(0.25)S(0) at 35°C (39.2 Pa, Tab. 4) is not enough to resist the deformation and it
532
takes a certain time to achieve enough mechanical strength after extruded onto deposited platform at room
533
temperature (25°C, tgel 86s, Fig. 5F). Secondly, Tgelation of materials obtained from rheological tests
534
closely related to the cooling rate (Gladkowska-Balewicz et al., 2014). The cooling rate of 3°C/min used
535
in the study might be resulting in an overestimation of Tgelation.
536
3.6.3 Three-dimensional structure (3D structure)
537
A hollow cylinder was printed to investigate inks’ ability to fabricate complex 3D structures (Fig. 9A).
538
The cylinders printed using ink kC(1)X(0.25)S(2) and kC(1)X(0.5)S(2) presented good shape retention
539
even under the load of many successive layers (19 layers), which is difficult to achieve using soft
540
hydrogel inks. Comparatively, the cylinder fabricated with ink kC(1)X(0.25)S(0) showed very poor shape
24
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541
retention and it collapsed dramatically during printing process. Several factors below may be accounted
542
for this fact. Firstly, Tgelation of ink kC(1)X(0.25)S(0) was 35.4°C (Tab. 4), obviously lower than the
543
printing temperature (40°C). As such condition, the ink was too liquid after extrusion and could not
544
rapidly recover enough mechanical strength to be able to self-support. Secondly, the ink
545
kC(1)X(0.25)S(0) with a longer tgel (89s) indicated that it would take longer to achieve gelation after
546
being extruded out, during which the extruded filaments flows continually until gelation occurred. Lastly,
547
the G of ink kC(1)X(0.25)S(0) was significantly lower than other inks with a value of 51.2 Pa at 10 rad/s
548
at 25°C. This poor mechanical strength might also exacerbate the deformation of printed cylinder. To
549
better characterize the deformation of cylinders, the parameter of wall thickness was used to evaluate
550
inks’ ability to fabricate complex 3D structures, as collapsing of cylinders would definitely increase it. As
551
seen from Fig. 9D, the wall thickness of cylinders printed with kC(1)X(0.25)S(0) and kC(1)X(0.5)S(0)
552
were significantly higher than that of cylinders fabricated with ink kC(1)X(0.25)S(2) and
553
kC(1)X(0.5)S(2). Again, the three factors described above might be accounted for this phenomenon,
554
namely Tgelation, gelation time tgel, and mechanical strength (G). Note that the wall thickness of
555
cylinders was larger than target thickness (3.5 mm), regardless of the used inks (Fig. 9D). Also note that
556
generally the width of extruded-out filament is bigger than theoretical value, while the height of filament
557
is lower, as a result of gravity and fusion between ink layers. As illustrated in Fig. 9B and Fig. 9C, the
558
schema of width difference (W) caused by first layer deformation and fusion between adjacent ink
559
layers definitely contributed to the larger wall thickness than desired value. Moreover, the wall thickness
560
deviation might also be related to the slicing software Skeinforge, which is specially designed for plastic
561
materials like polylactide, without any consideration for food materials and hydrogel properties.
562
4 Conclusions
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563
In this work, gel model multi-component systems composed of carrageenan, xanthan gum and starch
564
were prepared for an extrusion-based 3D food printer. The 3D printing process was divided into three
565
stages and corresponding rheological properties of inks for each stage were determined, namely extrusion
566
stage (yield stress, viscosity, shear-thinning), recovery stage (shear recovery, temperature recovery) and
567
self-supporting stage (G). Finally, printability of inks was systematically studied by printing 1D structure,
568
2D structure 3D structure. Addition of starch and xanthan gum increased inks’ Tgelation, viscosity, yield
569
stress, G*, and enhanced inks’ shear-thinning behaviour and reduced inks’ tgel. During extrusion stage,
570
inks’ extrudability was significantly affected by yield stress and shear-thinning behaviour. Tgelation, tgel
571
and G* are important for inks’ printability and shape retention performance. The insights achieved from
572
this study will provide guidance for food printing processes in which hydrocolloids are used as additives
573
to improve printing behaviour of the food formulations.
574
Acknowledgments
575
The authors acknowledge the financial support from the China State Key Laboratory of Food Science
576
and Technology Innovation Project (Contract No. SKLF-ZZA-201706), National First-class Discipline
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Program of Food Science and Technology (No. JUFSTR20180205), Jiangsu Province (China)
578
“Collaborative Innovation Center for Food Safety and Quality Control” Industry Development
579
Program, Jiangsu Province Key Laboratory Project of Advanced Food Manufacturing Equipment and
580
Technology (Contract No. FMZ201803), which have enabled us to carry out this study.
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Fig. 1 A: Research framework diagram. Tgelation, gelation temperature; PT, printing temperature; RT,
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room temperature. B: Photograph of the printer used in the study. C: Designed 43 mm43 mm lattice
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scaffold model with a 7 mm7 mm spacing between 3 mm fiber. D: Designed 15 mm height hollow
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cylinder with a 3.5 mm wall thickness and 25 mm inside diameter.
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Fig. 2 A: Temperature ramp tests with a cooling rate of 3°C/min for 2% starch (S(2)), 0.25% xanthan gum
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(X(0.25)), 1% k-carrageenan (kC(1)), and mixture system kC(1)X(0.25) and kC(1)X(0.25)S(2); B:
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Illustrative example of gelation temperature (Tgelation) for the composition kC(1)X(0.25)S(0) with a
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cooling rate of 3°C/min. Tgelation was determined by extrapolating the high and low temperature
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asymptotes of the viscosity and specifying the temperature at which these intersect. C: Illustrative
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example of yield stress for the composition kC(1)X(0.25)S(2) at 45°C. The yield stress of inks has been
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determined as the cross-over point where elastic modulus (G) equals to viscous modulus (G).
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Fig. 3 Viscosity and shear stress profiles for different inks at temperature of 35°C, 40°C and 45°C over
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shear rate of 0.01~100 1/s.
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Fig. 4 A: Recovery tests of inks conducted at 40°C under alteration of high (100 1/s) and low (1 1/s) shear
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rate. B: Shear recoverability of inks determined as the percentage of viscosity obtained during the first 30s
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in the third step after high shear rate (100 1/s) based on the average viscosity obtained in the first step.
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Fig. 5 A: Illustrative example of gelation time (tgel) of kC(1)X(0.5)S(0) through temperature change from
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printing temperature (PT, 40°C) to room temperature (RT: 25°C) indicated the response of complex
734
modulus (G*). B, C, D, and E: Gelation kinetics of kC(1)X(0.25)S(0), kC(1)X(0.5)S(0),
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kC(1)X(0.25)S(2), and kC(1)X(0.5)S(2) through temperature change from PT to RT indicated the
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response of G*. F: Gelation time (tgel) of different inks at different temperature.
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Fig. 6 Viscoelasticity properties of different inks within frequency of 1~100 rad/s conducted at room
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temperature 25°C. A: Elastic modulus. B: Viscous modulus. C: Complex modulus.
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Fig. 7 A: Line printing behaviour of ink kC(1)X(0.5)S(2) at printing temperature of 35°C, 40°C, 45°C and
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50°C. B: Schema of overlap (a) and printed pentagrams with different angles (b, c and d) using ink
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kC(1)X(0.25)S(2). C: Printed pentagrams using ink kC(1)X(0.25)S(2) at temperature of 35°C, 40°C, 45°C
748
and 50°C.
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751
752
Fig. 8 A: Images of printed scaffolds using different inks at different temperature. B: Schema of printed
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interconnected channel holes at different status. C: Average area of interconnected channels of printed
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scaffolds.
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Fig. 9 A: Images of hollow cylinders printed at 40°C viewed from different direction. B: Schema of width
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deformation (W) of first ink layer. C: Schema of width deformation of second ink layer and its fusion
760
with first layer. D: Wall thickness of hollow cylinders printed at 40°C using different inks.
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Tab. 1: Concentration levels of k-carrageenan, xanthan gum and potato starch applied by 23 factorial design
Independent variables
Lower level (-1)
Upper level (+1)
k-Carrageenan (%w/w)
1
2
0.25
0.5
0
2
Xanthan (%w/w)
Potato Starch (%w/w)
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767
768
Tab. 2: Experimental matrix designed for the independent variables concentration of k-carrageenan,
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xanthan gum and potato starch (%w/w) in water.
Coded values
k-Carrageenan
(%w/w)
Xanthan
(%w/w)
Potato Starch
(%w/w)
Ink Formulations
(-1, -1, +1)
(-1, -1, -1)
(-1, +1, +1)
(-1, +1, -1)
(+1, -1, +1)
(+1, -1, -1)
(+1, +1, +1)
(+1, +1, -1)
1
1
1
1
2
2
2
2
0.25
0.25
0.5
0.5
0.25
0.25
0.5
0.5
2
0
2
0
2
0
2
0
kC(1)X(0.25)S(2)
kC(1)X(0.25)S(0)
kC(1)X(0.5)S(2)
kC(1)X(0.5)S(0)
kC(2)X(0.25)S(2)
kC(2)X(0.25)S(0)
kC(2)X(0.5)S(2)
kC(2)X(0.5)S(0)
770
* In the codes listed for formulation column; kC, X and S stands for k-carrageenan, xanthan gum and
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potato starch, respectively.
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774
775
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Tab. 3 Gelation temperature (Tgelation), elastic modulus (G), viscous modulus (G) and complex modulus
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(G*) of different inks
Formulations
Tgelation(○C)
G (Pa)
G(Pa)
G* (Pa)
kC(1)X(0.25)S(0)*
35.4±0.3a
51.2±4.6a
39.4±1.3a
64.6±1.6a
kC (1)X(0.5)S(0)
35.7±0.1ab
377.5±19.6b
91.6±5.6b
388.4±10.2b
kC (1)X(0.25)S(2)
36.9±0.0bc
577.1±26.3c
123.2±4.9c
590.1±11.0c
kC (1)X(0.5)S(2)
38.2±0.1c
814.8±34.6e
189.8±16.3e
836.6±6.4e
kC (2)X(0.25)S(0)
44.2±0.9d
682.4±36.5d
133.3±18.6cd
694.3±13.3d
kC (2)X(0.5)S(0)
44.9±1.1d
831.0±16.9e
140.5±11.2d
842.8±14.6e
kC (2)X(0.25)S(2)
46.7±0.6e
1808.0±68.9f
260.5±4.9f
1827±21.2f
kC (2)X(0.5)S(2)
47.5±0.2e
9017.0±48.6g
1026.0±5.6g
9075±18.3g
778
* In the codes listed for formulation column; kC, X and S stands for k-carrageenan, xanthan gum and
779
potato starch, respectively. Values with different lowercase letters in the same column are significantly
780
different (p<0.05). G, G and G* were recorded at 10 rad/s.
781
782
783
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Tab. 4 Yield stress, consistency index (K) and flow index (n) of inks at different temperature
Formulations
Yield stress (Pa)
kC(1)X(0.25)S(0)*
35°C
39.24.3
40°C
#
45°C
#
kC(1)X(0.5)S(0)
35°C
80.27.1
40°C
#
45°C
#
kC(1)X(0.25)S(2)
35°C
134.23.7
40°C
13.70.6
45°C
12.00.5
kC(1)X(0.5)S(2)
35°C
551.324.0
40°C
60.53.1
45°C
36.91.8
K (Pa·sn)
n
R2
2.160.04
1.700.03
1.270.03
0.580.01
0.630.01
0.710.03
0.990
0.989
0.957
6.190.09
3.140.06
2.680.05
0.500.01
0.620.02
0.610.01
0.996
0.986
0.987
10.030.21 0.470.01
9.780.16 0.450.03
7.620.10 0.480.00
0.997
0.994
0.997
23.880.56 0.360.01
21.720.44 0.360.00
16.320.35 0.360.01
0.996
0.996
0.996
789
* In the codes listed for formulation column; kC, X and S stands for k-carrageenan, xanthan gum and
790
potato starch, respectively. In the column for yield stress, “#” indicates that the inks showed liquid character with
791
elastic modulus (G) smaller than viscous modulus (G) in the stress of 1~1000 Pa.
792
793
794
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Linking rheology and printability of a multicomponent gel system of
carrageenan-xanthan-starch in extrusion based additive manufacturing
Highlights
Thixotropic and thermo-reversible gel system was used as a model in 3D printing
Ink’s rheology and 3D printability was correlated in the whole printing process
1D, 2D, 3D structures were fabricated and correlated with ink’s rheology.
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