Exploration of the co-structuring and stabilising role of flaxseed gum in whey protein isolate based cryo-hydrogels

In the present work, the structuring and stabilising potential of flaxseed gum (FG) in whey protein isolate (WPI) cryo-hydrogels was investigated. The FG presence (0.1–1% wt.) in the heat-treated WPI dispersions (10% wt.) induced strong segregative phase separation phenomena, which were associated with a depletion flocculation mechanism.

Carbohydrate Polymers 289 (2022) 119424 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Exploration of the co-structuring and stabilising role of flaxseed gum in whey protein isolate based cryo-hydrogels Thierry Hellebois a, b, Claire Gaiani b, c, S´ ebastien Cambier a, Anaïs Noo a, Christos Soukoulis a, * a Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology (LIST), Avenue des Hauts Fourneaux, Esch-sur-Alzette, L4362, Luxembourg b Universit´e de Lorraine, LIBio, Nancy, France c Institut Universitaire de France (IUF), France A R T I C L E I N F O A B S T R A C T Keywords: Mucilage Cryotropic gelation Microstructure Phase separation Colloidal stability Molecular interactions In the present work, the structuring and stabilising potential of flaxseed gum (FG) in whey protein isolate (WPI) cryo-hydrogels was investigated The FG presence (0.1–1% wt.) in the heat-treated WPI dispersions (10% wt.) induced strong segregative phase separation phenomena, which were associated with a depletion flocculation mechanism The cryotropic processing of the WPI-FG solutions led to the formation of diverse macroporous protein gel networks depending on the colloidal state of their biopolymeric precursors Cryogel formation was primarily mediated via covalent (thiol-disulphide bond) bridging, whilst to a lesser extent, non-covalent in­ teractions contributed to the overall stabilisation of the protein gel network Although FG had a rather minor contribution to the formation of elastically active crosslinks (FG was partitioning mainly into the serum phase located in the macropores), its presence (at concentrations ≥0.75% wt.) improved the homogeneity and inter­ connectivity of the stranded protein network, whilst it reduced its colloidal instability and macroporosity Introduction Biobased cryogels constitute hydrogel templates produced via the cryogenic processing (i.e., freezing, incubation in the frozen state and thawing) induced crosslinking of biopolymer precursors (Lozinsky & Okay, 2014) Hitherto, polymeric precursors such as polysaccharides (e g galactomannans (Hellebois, Gaiani, et al., 2021; Lozinsky et al., 2000), glucomannans (Guo et al., 2022), glucans (Lazaridou & Bilia­ deris, 2004), xanthan (Giannouli & Morris, 2003), starch (Zou & Bud­ tova, 2020), sodium alginate (Gurikov & Smirnova, 2018) etc.) and proteins (e.g egg white (Balaji et al., 2019), whey protein isolate (Hellebois et al., 2022; Shiroodi et al., 2015), and gelatine (Regand & Goff, 2003; Savina et al., 2011) have been successfully employed in the development of biodegradable, biocompatible and mechanically rein­ forced cryogels It is well established that the cryogenic processing conditions (e.g ice nucleation and crystal growth, incubation temper­ ature and duration in the frozen state), the thawing rate, the amount and thermophysical profile of the polymeric precursors, as well as the presence of chaotropes or kosmotropes (e.g sugars, aminoacids, salts etc.) govern the mechanical and structural characteristics of the ob­ tained hydrogels (Lozinsky, 2020; Lozinsky & Okay, 2014) Cryo- hydrogels exhibit a tangible industrial relevance, including biomed­ ical, tissue engineering, biotechnological, environmental, pharmaceu­ tical, cosmetics and food applications In the latter case, cryo-hydrogels have been successfully implemented for structuring (active fillers), texturing and cryo-stabilising purposes (Coria-Hern´ andez et al., 2021; Patmore et al., 2003; Yang et al., 2020; Zhao et al., 2018) In addition, the feasibility of cryo-hydrogels as alternative biopolymer-based tem­ plates for controlled/sustained release of bioactive compounds has been showcased (Lazaridou et al., 2015; Nakagawa & Nishimoto, 2011; Sowasod et al., 2013) From a mechanistic standpoint, the cryotropic gelation may involve both non-covalent (e.g hydrogen bonds, van der Waals forces or hy­ drophobic interactions) and covalent interactions, resulting in physical (“reversible”) or permanent hydrogels (Gulrez et al., 2011; Lozinsky, 2018) For instance, galactomannans such as locust bean gum, alfalfa gum, and fenugreek gum are known to interact primarily via interchain polymer hydrogen bond bridging and intermolecular association of the smooth regions (i.e galactosyl depleted) via hydrophobic interactions (Dea et al., 1977; Hellebois, Gaiani, et al., 2021; Lozinsky et al., 2000) Similarly, oat β-glucans and xanthan gum based cryo-hydrogels are stabilized via -intra- and interchain hydrogen bonds in the junction * Corresponding author E-mail address: christos.soukoulis@list.lu (C Soukoulis) https://doi.org/10.1016/j.carbpol.2022.119424 Received 28 January 2022; Received in revised form 28 February 2022; Accepted 26 March 2022 Available online 29 March 2022 0144-8617/© 2022 Luxembourg Institute of Science and Technology Published by Elsevier Ltd (http://creativecommons.org/licenses/by/4.0/) This is an open access article under the CC BY license T Hellebois et al Carbohydrate Polymers 289 (2022) 119424 zones of the polymeric network (Giannouli & Morris, 2003; Lazaridou & Biliaderis, 2004) In the case of proteins, cryogenic processing induced gelation is driven by both covalent and non-covalent molecular in­ teractions For example, Hellebois et al (2022) reported that the addi­ tion of 25 mM of n-ethylmaleimide (NEM) hampered the ability of whey proteins to undergo cryogenic crosslinking, postulating that their cry­ ogelation ability is primarily associated with the occurrence of thioldisulphide (− SH/− S − S− ) bridging It is well documented that the cryogenic processing of heteropolymeric aqueous systems, i.e protein polysaccharide blends may offer tangible cryo-structuring and cryostabilising benefits For instance, the presence of soluble poly­ saccharides (i.e alfalfa galactomannan, xanthan/curdlan hydrogel complex) in denaturated whey protein solutions led to a significant enhancement of the mechanical strength and colloidal stability (Helle­ bois et al., 2022; Shiroodi et al., 2015) In a similar manner, Goff et al (1999) and Patmore et al (2003) demonstrated that locust bean gum exerts a strong cryostabilising role in model ice creams (sucrose + skim milk powder) due to its inherent cryogelling ability under dynamic freezing conditions Flaxseed gum, i.e a by-product of the flaxseed oil industry, has received much attention as an emerging hydrocolloid due to its tech­ nofunctional versatility, environmental sustainability, low cost, as well as high biocompatibility and biodegradability (Liu et al., 2018) Although the chemical composition and structure conformational properties of flaxseed gum can vary on its genotypic and phenotypic characteristics, the extraction, isolation and purification conditions are also well known for impacting its proximate and osidic composition and consecutively, its technofunctional profile (Hellebois, Fortuin, et al., 2021; Liu et al., 2018; Liu et al., 2021; Soukoulis et al., 2018) Hitherto, flaxseed gum has been successfully employed in producing food com­ posites due to its prevalent thickening and gelling capacity (Liu et al., 2018; Soukoulis et al., 2018) In protein-rich hydrogel-based food sys­ tems, the stabilising, structurising and texturising effectiveness of flax­ seed gum depends on its colloidal interactions with proteins as influenced by extrinsic factors such as the ionic strength, pH and tem­ perature (Basiri et al., 2018; Chen et al., 2016; Kuhn et al., 2011; Li et al., 2012; Liu et al., 2018; Soukoulis et al., 2019) For example, the extent of segregative phase separation in flaxseed gum - whey protein binary systems is inextricably associated with the mechanical properties and the physical stability of the obtained cold or acid induced gels (Kuhn et al., 2011; Soukoulis et al., 2019) Li et al (2012) reported that the presence of flaxseed gum in cold-set casein gels resulted in the propor­ tional elevation of the onset gelation temperature due to the adsorption of flaxseed gum onto the micelles, facilitating their intermolecular bridging In a previous work, Hellebois et al (2022) demonstrated that the presence of alfalfa gum, i.e., a non-ionic galactomannan extracted from the endosperm of alfalfa seeds (Hellebois, Gaiani, et al., 2021), in whey protein cryogels possess an active filler mediating role due to its inherent ability to form soft cryogels This was associated with a remarkable improvement of the mechanical properties and colloidal stability of the whey protein cryogels (Hellebois et al., 2022) In the present work we aimed at exploring the functional role of an anionic natural poly­ saccharide, i.e flaxseed gum in the cryo-gelling performance of whey protein isolate Hereby, the fundamental question is whether and to which extent flaxseed gum can mediate the cryogelation mechanistic action of whey proteins, and hence, the structural conformation, me­ chanical and physical properties of the obtained hydrogels minerals 2.5%) was kindly donated by Ingredia (Arras, France) The flaxseed gum (FG) was extracted at mild alkaline conditions (pH = 8) from golden flaxseeds and fully characterised as detailed in Hellebois, Gaiani, et al (2021) The FG was composed of 87.1% carbohydrates (8.4% arabinose, 25.3% xylose, 24.2% rhamnose, 22.3% galacturonic acid, 14.3% galactose, 4.2% fucose and 1.4% glucose), 7.2% proteins and 5.7% ash, on a dry basis, while lipids were detected in traces The polysaccharidic populations corresponded to arabinoxylans (AX), rhamnogalacturonan–I (RG-I), and two AX-RG-I composite fractions The gum extract exhibited a molecular weight of 1.3 × 106 Da, an intrinsic viscosity of 6.52 dL g− 1, a critical coil overlap concentration (c*) of 0.55% and an average z-diameter of 97.8 nm All the chemicals used were purchased from Sigma Aldrich (Leuven, Belgium), and they were of analytical grade 2.2 Preparation of the WPI-FG solutions and cryogels WPI aqueous aliquots (10% wt in protein matter) were prepared by dispersing ca 11.65 g 100 g− of WPI powder into Milli-Q water (18 mΩ, Merck-Millipore Inc., Burlington, US) and under gentle mechanical stirring (IKA GmbH, Staufen, Germany) overnight to allow complete hydration of the whey proteins The pH of the WPI solutions was adjusted at 7.00 ± 0.05 using NaOH M The insoluble particle impu­ rities were removed ( 0, which in­ dicates a synergistic interaction between the biopolymers in terms of apparent viscosity It is well known that the steady state and oscillatory rheological behaviour of biopolymer aqueous systems is associated with To understand the cryo-hydrogel formation performance of WPI as influenced by the presence of FG, the elastic modulus G′ and particle size (d4,3) values of the WPI-FG systems were recorded for five consecutive T Hellebois et al Carbohydrate Polymers 289 (2022) 119424 Fig Flow behaviour curves (A), double logarithmic plot of the viscosity vs concentration (B) and biopolymer interaction coefficient R (C) of WPI solutions as influenced by FG concentrations (0.1–1% wt.) a-dDifferent letters between bars denote a significant difference (p < 0.05) freeze-thaw cycle events between 25 and − 28 ◦ C (Fig 4) In keeping with our previous findings (Hellebois et al., 2022), the sequential freezethaw processing of the WPI-FG solutions led to a progressive increase in the G′ values After a single freeze-thaw event, a significant improve­ ment of the elastic modulus of the WPI-FG systems − proportionally to cFG− was observed The cryogenic structuring was kept improving until the end of the second freeze-thaw event and then, a pseudo-equilibrium viscoelastic state was achieved (Fig 4A) It should be pointed out that the reinforcement of the elasticity of the formed cryogels was adversely associated with the colloidal instability index of the polymeric precursor systems (r = − 0.695, p < 0.05) Contrarily to the WPI – alfalfa gum systems (Hellebois et al., 2022), which exerted a cycle-by-cycle structuring effect owing to the concomitant cryogelling activity of the biopolymer precursors, in the case of WPI-FG systems it was not possible to identify any clear contribution of FG in the cryogelation process The negligible cryogelation ability of FG was also confirmed when individual FG solutions (c* = 0.55% < cFG < 5% wt.) were cryogenically processed at the same conditions (data not shown) Hence, it is dictated that FG contributes to the viscoelastic build-up of the cryogels probably via an inert filler role partitioning as a major solute of the cryo-concentrated serum phase (see also section 3.3) Yet, the increasing concentration of FG did not impair (0.1 ≤ cFG ≤ 0.75% wt.) or even led to an improvement (at cFG = 1% wt.) of the elasticity of the final cryohydrogels Mixed effects as concerns the impact of the polysaccharidic Fig Dynamic changes in the elastic modulus (A) and mean particle size (B) of WPI-FG solutions occurring throughout cryogenic processing between 25 and − 28 ◦ C a-f, A-CDifferent letters between concentrations (lowercase) or among the cycles (uppercase) indicate a significant difference (p < 0.05) T Hellebois et al Carbohydrate Polymers 289 (2022) 119424 precursor on the G′ values of WPI based cryogels have been previously reported for galactomannans (i.e active filler effect), (Hellebois et al., 2022) and xanthan/curdlan co-polymer blend (i.e adverse effect) (Shiroodi et al., 2015) beyond a specific concentration have been reported In Fig the microstructural features of the WPI-FG cryogels following a single and five consecutive freeze-thaw cycles are illus­ trated As seen in the CLSM acquired micrographs, cryogenic processing of the WPI-FG precursor solutions led to the formation of macroporous hydrogel templates consisting of irregularly interconnected stranded WPI-based structures Interestingly, the hydrogel templates developed in the end of the first freeze-thaw event retained partially the micro­ phase separated structure conformation of the unprocessed polymeric solutions; this was fairly evidenced in the case of the bicontinuous WPIFG solution (Fig 5–0.25% wt.) Likewise, the presence of the clustered whey protein aggregates was evidently seen in the case of the cryogels comprising at least 0.75% of FG The ability of arresting the micro­ structure of segregated biopolymer systems via sol-gel (cold, heat or acid induced) or rubbery to glass physical state transitions is well-reported (Tanaka, 2012; Turgeon et al., 2003) Throughout freeze-thaw cycling, a re-organisation of the cryogels microstructure was observed as represented by an overall increase in the thickness of the protein strands and the macropores mean size This behaviour is suggestive of the inertness of FG as cryostructuring or cryostabilising polymeric precursor Indeed, it was recently shown that when a cryogel forming polymeric precursor was added into the WPI dispersions, no significant changes in the microstructural elements of the cryogels could be detected throughout the cryostructuration process (Hellebois et al., 2022) It is well documented that the cryotropically induced hydrogels may be obtained via either physical (non-covalent) or chemical (covalent) crosslinking (Lozinsky, 2018, 2020; Lozinsky & Okay, 2014) As for food biopolymers, i.e polysaccharides or proteins, cryogelation may occur via different mechanistic pathways In general, polysaccharide based cryogels are primarily of physical character i.e the cryogelation process is triggered through weak interchain polymer crosslinking owing to hydrogen bonding and hydrophobic interactions (Giannouli & Morris, 2003; Hellebois, Gaiani, et al., 2021; Lazaridou & Biliaderis, 2004; Lozinsky et al., 2000; Tanaka et al., 1998) On the other hand, food proteins e.g milk proteins, egg white or gelatine can be formed on the basis of concomitant non-covalent and covalent (mainly disulphide bonding) interactions (Balaji et al., 2019; Hellebois et al., 2022; Savina Fig CLSM micrographs of the WPI-FG cryogel obtained after one and five freeze-thaw cycles influenced by the presence of FG (0–1% wt.) Scale bar = 50 μm T Hellebois et al Carbohydrate Polymers 289 (2022) 119424 et al., 2011) To get a better overview of the mechanistic background of the cry­ ostructuration of the WPI-FG systems, the obtained hydrogels at the end of the freeze-thaw process were dissolved in different solvents aiming at cleaving specific biopolymer molecular interactions such as: a) hydrogen bonding and electrostatic complexation (phosphate buffer), b) hydrophobic interactions (SDS containing phosphate buffer), and c) covalent bonding (DTT/SDS containing phosphate buffer (Tanger et al., 2021) As illustrated in Fig 6, the cryostructuration of the WPI-FG so­ lutions was governed by thiol-disulphide (− SH/− S − S− ) interchange interactions favoured by the heat induced dissociation and unfolding of the native whey protein molecules and the increased molecular mobility of the peptides (Nicolai et al., 2011) To a lesser extent, nonspecific (i.e electrostatic and hydrogen bond) and hydrophobic interactions, ac­ counting for approx 20 and 3% of the total protein solubility, respec­ tively, also contributed to the cryo-hydrogel formation (Fig 6) As concerns the contribution of FG to the cryotropically mediated molec­ ular interactions, a proportional to FG content dependence of the nonspecific interactions (r = − 0.856, p < 0.001) and disulphide bridging (r = 0.837, p < 0.001), respectively, was identified As urea could not be tested as a hydrogen bond blocking agent (due to method restrictions) and taking into account the minor contribution of FG to the overall surface charge density, hereby it is hypothesised that the nonspecific interactions represent mainly hydrogen bond bridging In this context, it appears that the reduction of the hydrogen bonding prevalence on FG addition stemmed from the segregative phase separation of the bio­ polymers The latter hindered partially the protein – polysaccharide hydrogen bond interactions, whilst at the same time arose the − SH/− S − S− interchain protein crosslinking (r = − 0.908, p < 0.001) On the other hand, the contribution of FG to the prevalence of the hydrophobic interactions remained unclear Similar effects have been also reported in the case of microphase separated WPI-polysaccharide heat-set hydrogels (Zhang et al., 2021) Table Viscoelastic properties of the WPI-FG cryogels obtained after five freeze-thaw cycles Gum content Amplitude sweeps (% wt.) (Pa) (%) (Pa) (Pa) 9.5 ± 0.7a 12.5 ± 0.2a 8.0 ± 1.5a 12.2 ± 2.5a 24.0 ± 4.8b 26.9 ± 1.9b 1.03 ± 0.09a 1.29 ± 0.12a 1.17 ± 0.21a 1.22 ± 0.18a 1.29 ± 0.12a 1.43 ± 0.00a 109.4 ± 20.5a 113.8 ± 1.6a 58.4 ± 0.5a 101.7 ± 7.6a 191.6 ± 61.6b 230.3 ± 61.9b 80.3 ± 13.5a 88.4 ± 8.2a 66.1 ± 9.9a 96.4 ± 8.3a 200.3 ± 35.3b 227.1 ± 5.5b 0.1 0.25 0.5 0.75 τy γ˙ LVE Frequency sweeps τf G'f G'-f slope tanδ at Hz 0.081 ± 0.050a 0.093 ± 0.006ab 0.096 ± 0.009b 0.103 ± 0.003bc 0.109 ± 0.005cd 0.119 ± 0.006d 0.169 ± 0.009a 0.175 ± 0.008a 0.185 ± 0.006ab 0.199 ± 0.010bc 0.217 ± 0.013cd 0.236 ± 0.007d a-d Different letters between concentrations for each rheological property indi­ cate a significant difference (p < 0.05) Abbreviations used: γ˙ LVE : strain at the end of the LVE boundary; τy: yield stress; τf: flow stress; G'f: Storage modulus at flow stress Measurements performed at 25 ◦ C, Hz for amplitude sweeps and 0.1% strain for frequency sweeps (Fig 7) and colloidal instability (Fig 8) characteristics For mapping the mechanical profile of obtained cryogels, the normalised elastic modulus (i.e G′ /G′ 0) strain sweep rheological spectra were assessed (Suppl Fig 1A) According to Ross-Murphy (1995), the strain dependence of the reduced modulus can be used to distinguish weak from strong gels As illustrated in Suppl Fig 1A and Table 2, the G′ /G′ values started to ˙ become strain dependent at γ≫0.05% that is suggestive of a strong gel The yield point τy (i.e., the minimum required stress to induce irre­ versible internal structure conformational changes) values were signif­ icantly increased only when cFG ≥ 0.75% wt Further increase in the deformation stress (at τf) resulted in the structural collapse of the cry­ ogels as result of the increasing molecular motion of the polymeric moieties that are not strongly fixated in the whey protein gel network However, only when cFG exceeded 0.75% wt., a significant increase in the flow point values could be observed The strain sweep data confirm the hypothesis that FG does not possess an active filler role; instead, it acted as a thickener of the serum phase present in the cryogel macropores To further investigate their frequency dependent behaviour, the WPI-FG cryogels were subjected to frequency sweep tests (Suppl Fig 1B) Fitting the elastic modulus – frequency data to the power model ′ i.e., G′ = K′ ωn allowed the calculation of the slopes of the rheological spectra As given in Table 2, the slopes ranged from 0.08 to 0.12 indi­ cating the formation of strong physical gels that involve covalent crosslinking The obtained values are generally in keeping with the literature data regarding cold or acid protein gels comprising flaxseed gum (Chen et al., 2016; Kuhn et al., 2011; Soukoulis et al., 2019) The progressive increase in the n' parameter and damping factor (tanδ) values implies that the FG presence in the WPI cryo-hydrogels amplified their viscoelastic character Contrary to galactomannans, which exert an active filler activity endowing composite-like characteristics to the WPI cryogels (Hellebois et al., 2022), the FG did not improve remarkably the elastic component of the cryo-hydrogels due to its inexistent cryogelling ability For gaining a better insight into the structural elements of the WPIFG cryogels, the obtained CLSM micrographs at the end of the freezethaw processing were subjected to image analysis using the AngioTool software adopting the computational analysis protocol described in detail by (Zudaire et al., 2011) In Fig 7A,B is given a schematic rep­ resentation of the CLSM acquired and the AngioTool computed micro­ structural conformation images of the WPI-FG cryogels Selected 3.3 Characterisation of the final WPI-FG cryogels The cryo-hydrogels obtained at the end of the 5th freeze-thaw event were assessed for their viscoelastic (Table 2), structural conformational Fig Normalised protein solubility indicative of the occurrence of noncovalent and covalent molecular interactions quantified in the WPI-FG cry­ ogels obtained in the end of the cryogenic processing a-dDifferent letters be­ tween bars (within the same type of molecular interaction) denote a significant difference (p < 0.05) T Hellebois et al Carbohydrate Polymers 289 (2022) 119424 Fig Indicative CLSM micrograph WPI cryogel obtained after five freeze-thaw cycles (A) and its analysed protein network using the AngioTool soft­ ware (B) The blue dots and red vessels represent the network junctions and skeleton, respectively The obtained macropore area occupancy is depicted in (C) and lacunarity in (D) a-cDifferent letters between concentrations for each stage indicate a significant difference (p < 0.05) (For interpretation of the ref­ erences to colour in this figure legend, the reader is referred to the web version of this article.) parameters such as the macropores space occupancy (Fig 7C) and the lacunarity (Fig 7D) were calculated As clearly depicted in Fig 7C,D the macroporosity and the heterogeneity (lacunarity) of the obtained cry­ ogels were positively (r = 0.651, p < 0.05; r = 0.679, p < 0.05, respectively) associated with the colloidal instability of the bio­ polymeric precursor system Thus, the hydrogels obtained from the cryogenic processing of bicontinuous or smaller interconnected phase separated systems were found as having a predominantly heterogenic macroporous structure Finally, it should be noted that at least 1% wt of FG is required for attaining a significant improvement of the structural uniformity and protein strands space occupancy On this occasion a significant increase (p < 0.001) in the number of the vessel intercon­ nection (junction) points was observed (Suppl Fig 2) One of the major challenges in the design of mechanically and physically resilient biopolymer cryogels is to prevent excessive solvent losses due to the structural re-organisation of the gel during the ageing process (Lucey, 2002) In general, milk protein gels are prone to syn­ eresis, that is the spontaneous exudation of the loosely held water found in the interspace of the entangled protein network (Lucey, 2020) It is well documented that parameters such as the coarseness, permeability and stiffness of the protein gel network are major determinants of the syneresis of milk protein gels (Urbonaite et al., 2015, 2016) To avoid any bias in the computed syneresis kinetics due to the mechanical disturbance of the hydrogel, aliquots of WPI-FG solutions (ca 1.8 mL) were transferred into the LUMiSizer cuvettes and processed cryogeni­ cally as above mentioned As illustrated in Fig 8, all cryo-hydrogels exhibited significant syneresis rates ranging from 7.5 to 9.1 μm s− Although there were no significant differences in the syneresis rates of the cryogels containing up to 0.5% wt of FG, the exuded serums had distinctively different turbidity The quantification of the residual pro­ tein in the serum samples, revealed a progressively eminent reduction in the proteinaceous matter content as function of cFG Similar behaviour has been reported in WPI heat-set gels containing acidic hetero­ polysaccharides (He et al., 2021) It is assumed that the residual protein partitioning in the serum dictates a lower ability of the proteins to un­ dergo disulphide bonding Interestingly, the rheological characterisa­ tion of the FG containing serum and the individual FG solutions (WPI free) failed to unveil any significant differences in terms of their apparent viscosity (Fig 8C) This gives support to our hypothesis that FG is found primarily as solute in the serum and therefore, it is ability to enable the formation of elastically active network chains, as previously showcased for FG-WPI acid gels (Soukoulis et al., 2019), is limited Conclusions The co-structuring and stabilising role of flaxseed gum in cryo­ tropically produced whey protein isolate hydrogels was studied Flax­ seed gum exhibited a strong thermodynamic incompatibility with whey proteins resulting into water-in-water emulsion like, bicontinuous, interconnected or aggregated conformational states depending on the protein to polysaccharide content ratios The segregative phase sepa­ ration phenomena were primarily ascribed to a depletion flocculation mechanism The cryogenic processing of the WPI-FG solutions resulted in the formation of macroporous hydrogels From a molecular stand­ point, the cryotropic gelation was mediated primarily via covalent interchain protein crosslinking (cysteine thiol-disulphide bridging) and to a lesser extent via non-covalent (i.e hydrogen bonds and hydropho­ bic) polymeric interactions Although FG had a modulating role on the prevalence of the molecular interactions, it did not arrest the micro­ structural re-organisation of the protein gel network during cryogenic processing This was attributed to the inability of FG to undergo cryo­ tropic gelation and therefore, to enact an active filler role within the fixated protein gel network The presence of FG at concentrations ≥0.75% wt improved the gel homogeneity and interconnectivity of the whey protein strands while it reduced the macropores mean size Hence, at least 1% wt of FG is required for constructing WPI cryo-hydrogels that could endow tangible structuring, stabilising and texturing bene­ fits to complex food matrices T Hellebois et al Carbohydrate Polymers 289 (2022) 119424 Fig LUMiSizer transmission spectra and macroscopic imaging of the 5-fold cycled WPI-FG cryogels after 30 of centrifugation at 2300g, 25 ◦ C and syneresis rate (A), protein content in the exuded serum (B) and viscosity of the exuded serum compared to FG (WPI free) solutions (dashed bars) (C) a-dDifferent letters between the bars indicate a significant difference (p < 0.05) CRediT authorship contribution statement Acknowledgement T Hellebois: Conceptualisation, Investigation, Formal Analysis, Writing Original Draft, Writing – Review and Editing S Cambier: Investigation, Writing – Review and Editing A Noo: Investigation, Resources C Gaiani: Writing - Review and Editing, Project administration, PhD student (TH) supervision C Soukoulis: Conceptualisation, Writing - Review and Editing, PhD student (TH) supervision, Project administration, Funding Acquisition This work was supported by the Luxembourg National Research Fund (FNR) (Project PROCEED: CORE/2018/SR/12675439) Mrs Manon Hiolle (Ingredia SA, France) is thanked for generously providing the whey protein isolate Appendix A Supplementary data Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2022.119424 Declaration of competing interest References The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Agoda-Tandjawa, G., Durand, S., Gaillard, C., Garnier, C., & Doublier, J.-L (2012) Rheological behaviour and microstructure of microfibrillated cellulose suspensions/ 10 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properties of starch aerogels and cryogels via starch source and process parameter Carbohydrate Polymers, 117344 https://doi.org/10.1016/j.carbpol.2020.117344 Zudaire, E., Gambardella, L., Kurcz, C., & Vermeren, S (2011) A computational tool for quantitative analysis of vascular networks PLoS ONE, 6(11), Article e27385 https:// doi.org/10.1371/journal.pone.0027385 12 ... leading to the increase in the protein aggregates mean size This finds also support on the dynamic light scattering measurements of the individual and binary biopolymer solutions showing that the. .. whilst at the same time arose the − SH/− S − S− interchain protein crosslinking (r = − 0.908, p < 0.001) On the other hand, the contribution of FG to the prevalence of the hydrophobic interactions... filler role within the fixated protein gel network The presence of FG at concentrations ≥0.75% wt improved the gel homogeneity and interconnectivity of the whey protein strands while it reduced the