Quality improvement of plant-based meat alternatives by addition of iota carrageenan to pea protein–wheat gluten blend

In this study, the influence of iota carrageenan (IC) addition at different steps to the protein blends based on pea protein isolate (PPI) and wheat gluten (WG) as well as hydration mixing time and temperature of IC on the quality attributes of plant-based meat alternatives was studied. In more detail, IC was added before (B, in water with mixing times of 15 or 30 min and temperatures of 25 or 75 °C) or after (A, in powder form) the addition of PPI to the mixture with or without calcium chloride (Ca) in the formulation. The results showed that the addition of IC after PPI, especially combination with Ca resulted in the products with the most visible fibers, which can be considered as a quality improvement. IC addition to the formulations with or without Ca also increased the browning index, water holding capacity, tensile stress, and air bubble numbers compared to the PPI.WG formulation. However, no considerable difference in these parameters was found regarding the addition order of IC (before or after the addition of PPI). As the addition of IC after PPI hydration needs less energy for mixing, and, thus, less time for preparation, this order of addition can be recommended for improving the quality of plant-based meat alternatives containing IC. Therefore, hydration of IC in water, especially at high temperatures, is not necessary for the production of plant-based meat alternatives produced in the high-temperature shear cell (HTSC).


Introduction
Julia Twigg, a sociologist, believes that "Meat is the most highly prized of food. It is the center around which a meal is arranged" [13]. Therefore, it is predictable that many people like to have meat in their diets. However, amongst others the increase of the world population to 9.1 billion by 2050 will lead to a doubling of the demand for meat if suitable plantbased meat alternatives are not found [8]. Plant-based meat alternatives can be defined as food products similar to meat produced from plant-based ingredients to mimic the appearance, texture, and nutritional value of real meat products [28]. Currently, a noteworthy growth of plant-based meat alternatives to traditional animal proteins has been in the food market [34]. Thus, different plant-based meat alternatives can be found in the market to provide our diets with enough protein [35] which can help to mitigate consumer's concerns about their health as well as the environmental and ethical aspects. The consumption of plant-based meat alternatives can decrease body weight, blood pressure, saturated fatty acids, and cholesterol resulting in a reduction in cancers, cardiovascular disease, stroke risk, diabetes, and mortality [2,28]. Additionally, hormone usage for faster and higher meat production [35] and excessive antibiotics used to fight new infections produced by deadly pathogens are other health concerns related to meat consumption [9]. Ethically, the huge demand for meat in the near future also increases concerns about animal welfare. Moreover, it results in numerous environmental problems such as natural resource depletion [32], deforestation, pollution, damage to hydrogeological reserves, and loss of biodiversity [9] that negatively affect climate change [32].
It is often suggested that plant-based meat alternatives should have similar textural, sensorial, and nutritional properties at an affordable cost to convince many consumers [10]. Currently, soy protein isolate is mostly used to produce plant-based meat alternatives [6,36]. However, soy is connected to GMO concerns and is allergenic. Furthermore, 1 3 soy is mostly produced in South American countries, and, thus, its transportation to Europe could negatively affect the environment. Moreover, soil erosion and severe logging for soy cultivation harmfully impact the environment. These concerns explain a rising interest in pea as ingredient for meat alternatives. Pea can be cultivated in Europe, which solves the transport emission problem. In fact, regional production of products has different advantages in resource use and environment and climate protection [39]. In addition, pea protein isolate (PPI) has gained interest due to its low allergenicity, reasonable cost, its suitability for production in temperate climates, and acceptable nutritional value [20]. Although pea protein contains high levels of essential amino acids, especially lysine, it is low in sulfur-containing amino acids, especially methionine and cysteine [20,24]. However, the combination of pea protein with cereal proteins such as wheat gluten (WG) can compensate for this problem as cereal proteins have high levels of sulfur amino acids. Thus, the mixture of PPI and WG has a balanced essential amino acid profile [20]. Thus, despite the fact that the development of plant-based meat alternatives containing PPI instead of soy protein isolate with appropriate textural and sensorial attributes is more challenging, PPI will probably be more easily accepted by consumers [31,33].
Hydrocolloids are generally used in foods to obtain desired textural and sensorial properties [9] with their ability to retain water and form gels. Carrageenan is generally obtained from red seaweeds [27]. It is composed of D-galactose units in three primary forms (kappa, iota, and lambda) [47]. Those units differ with respect to the quantity and location of sulfate groups on the galactose chain. The water solubility increases with iota carrageenan (IC) which contains two sulfate groups per two galactose units [17]. Some studies found positive effects on the texture and firmness of plant-based meat alternatives and fiber formation in these products by adding IC to soy protein concentrate [25]. However, no research has been done yet on the effect of IC in plant-based meat alternatives based on PPI and WG in the high-temperature shear cell (HTSC). Furthermore, little attention has been paid to the appropriate moment of addition of IC into the formulation as well as to the temperature of the mixture at the moment of addition and the time allowed for hydration. IC is dissolves better and more quickly at higher temperatures [7]. It is not even clear whether hydration of IC in water is necessary to obtain good structuring, but it is reasonable that the hydration time and temperature will be of influence. A limitation of the extruder is that powder should simultaneously be mixed with water in the system and thus, the order of addition and degree of hydration cannot be tested. However, as the protein mixture is prepared outside HTSC, these parameters can be investigated. Additionally, IC constructs elastic gel networks and thermoreversible gels with calcium ions [1]. Therefore, salts will also influence the quality attributes of plant-based meat alternatives.
This study, therefore, aims to investigate the influence of IC on the structuring of plant-based meat alternatives in a HTSC process by measuring the textural and the properties related to sensorial profile of plant-based meat alternatives based on PPI and WG. The hypothesis we follow is that the formulation and the procedure of IC addition to the protein blends influence the morphology of plant-based meat alternatives. For instance, a higher water holding capacity and gel strength can be obtained with a blend of protein and IC [25,42], and when IC should be hydrated, its hydration time and temperature will affect the structure of the processed materials obtained. Finally, the addition of Ca with IC in the product formulation improves the fibrous structure of plantbased meat alternatives and forms stronger gels [21,42,46].

Preparation of different protein blends
Regarding the abbreviations of the product names in Table 1, the letters on the left side and before the comma show the ingredients containing pea protein isolate (PPI), wheat gluten (WG), iota carrageenan (IC), and calcium chloride (Ca) in the formulations. Moreover, the letters and numbers after the comma show the IC addition procedure (after (A) PPI addition and in powder form to the protein mixture or before (B) PPI addition to the water with IC hydration temperatures of 25 °C or 75 °C and IC hydration times of 15 min or 30 min). All formulations described here were based on 35 wt% ingredients, with equal weights used for PPI and WG. These procedures for preparing of different protein blends (100 g) are described in more detail in the following Sects. ("Preparation of protein blend (PPI.WG)", "PPI.WG.Ca product", "PPI.WG.IC,A product", "PPI. WG.IC,B25°C-15min and PPI.WG.IC,B25°C-30min products", "PPI.WG.IC,B75°C-15min and PPI.WG.IC,B75°C-30min products", "PPI.WG.IC.Ca,B25°C-15min and PPI.WG.IC.Ca,B25°C-30min products", "PPI.WG.IC. Ca,B75°C-15min and PPI.WG.IC.Ca,B75°C-30min products", "PPI.WG.IC.Ca,A product").

Preparation of protein blend (PPI.WG)
The control without IC was denominated as PPI.WG in Table 1 and contained pea protein isolate (PPI) and wheat gluten (WG). First, PPI (17.5 g) and demineralized water (65 g, room temperature) were mixed at room temperature by hand with a spatula for approximately 30 s in a beaker. Then, the beaker was covered with parafilm to prevent water evaporation and left for PPI hydration at room temperature for 30 min. Next, WG (17.5 g) was added to the mixture and mixed manually with the spatula. Finally, the blend was processed in the HTSC (Fig. 1).

PPI.WG.Ca product
To evaluate the effects of calcium chloride (Ca) in the product containing PPI and WG, Ca (0.4%) was added to the formulation. PPI (17.3 g) and Ca (0.4 g) were mixed together and added to a beaker with demineralized water (65 g). Then, all the mentioned ingredients were mixed manually with the spatula for approximately 30 s. Next, the beaker was covered with parafilm and left for hydration at room temperature. After 30 min, WG (17.3 g) was added to the hydrated mixture and was mixed with the spatula (Fig. 1).
PPI.WG.IC,A product PPI.WG.IC,A contains PPI, WG, and IC with the addition of iota carrageenan (IC) after (A) PPI addition. Similar to standard procedure, PPI (16.5 g) and demineralized water (65 g) were mixed manually. After hydration for 30 min, WG (16.5 g) and IC (2 g) were mixed together and added to the mixture. Next, they were mixed by hand with the spatula (Fig. 1). WG.IC,B25°C-30 min samples, respectively with a stirrer (EUROSTAR 40 digital, IKA, Germany) and a four-flatblade turbine impeller at 400 rpm. After mixing, the mixture was weighed to determine the amount of water that evaporated, and demineralized water was added to compensate for the loss. Then, PPI (16.5 g) was added to this PPI.WG.IC.Ca,A product PPI.WG.IC.Ca,A sample contains PPI, WG, Ca, and IC with the addition of IC after (A) hydration of PPI in the mixture. First, PPI (16.3 g) and Ca (0.4 g) were mixed together in the beaker with demineralized water (65 g) with a spatula for approximately 30 s. After 30-min hydration at room temperature, WG (16.3 g) and IC (2 g) were added to the protein blend and mixed manually.

High-temperature shear cell (HTSC) processing
Approximately 95 g of the described blends in Sects. "Preparation of different protein blends" and Table 1 were processed into a pre-heated (at 100 °C) HTSC designed at Wageningen University (The Netherlands). The shearing speed rotation for processing of all the formulations was 30 rpm for 15 min, and the processing temperature was at 100 °C. After 15 min of thermomechanical treatment, the device was cooled down to 25 °C for 10 min without any shearing (0 rpm). After cooling, the products were taken from the HTSC and placed in a resealable zipper bag at room temperature for at least 1 h before color and texture analyses and fiber observation. Then, the samples were stored at − 18 °C to continue with the subsequent analysis (SEM, CLSM, WHC, and XRT).

Visual inspection of fibrousness
Visualization of the macrostructure of the sheared samples was conducted by taking pictures inside an in-house white mini photo studio with three lights on the right, left, and upper sides. A picture of the complete products processed in the HTSC was taken inside a photo studio with standardized lighting. The macrostructure and fibrous structure of the products were visually evaluated by cutting and folding three pieces (Fig. 2) from products parallel to the shear-flow direction. Then, the cut pieces were collocated in a long pin for taking pictures via an iPhone 12, Apple.

Color measurement
A colorimeter (Chroma Meters CR-400, KONICA MINOLTA, INC., Japan) was used to measure the L*, a*, and b* color data of samples. L* is associated with lightness (minimum value of 0) and darkness (maximum value of 100), a* with redness (positive values) and greenness (negative values), and b* with yellowness (positive values) and blueness (negative values). Browning index (BI) was calculated using L*, a*, and b* data by Eq. (1) [40]:

Tensile strength analysis
The mechanical characteristics of the products were evaluated with a Texture analyzer (TA.XTplusC, Stable Micro Systems, UK) and based on the procedure described by Taghian Dinani, van der Harst, et al. [39] and Schlangen et al. [29]. Specimens were cut out of the sample with a Finally, Young's modulus [Pa] was calculated from the slope of the linear part of the tensile stress versus the tensile strain curve.

Water holding capacity (WHC)
For the WHC test, the procedure described by Taghian Dinani, Broekema, et al. [36] was followed. Three specimens were taken out with a ring mold of approximately 16 mm in diameter per replication. These three circular pieces were weighed and placed in a beaker with 70 ml of demineralized water, and the beakers placed in a water bath (SW23, JULABO, Germany) at 50 °C for 16-17 h. After that, the hydrated samples were carefully taken out and placed on a tube rack; surface water was carefully and quickly removed with tissue paper. Finally, the hydrated samples were weighted, and WHC was calculated using Eq. 4: In this equation W a and W b correspond to the weight of samples after and before hydration, respectively.

Confocal laser scanning microscopy (CLSM)
A similar procedure as described by Jia et al. [15] was followed to prepare CLSM images. First, frozen samples were cut in a right trapezoid shape (8 mm × 10 mm × 15 mm) parallel to the shear-flow direction. Then, the cut samples were glued with a CryoCompound (Immunologic a WellMed company, Duiven, The Netherlands) on a 35 mm cryostat chuck and collocated in a SLEF Cryostat MEV (SLEE, Mainz, Germany) to create slices with a smooth surface and with approximately 60 µm thickness at − 17 °C. The specimens were collocated on a microscope slide and stained with Rhodamine B (0.002% in MilliQ water). Consequently, the stained samples on the microscope slides were stored in a dark and humid box to maintain their moisture content constant at room temperature for at least 1 h. The samples were then confined by placing a coverslip. So-called spacers were used to create the appropriate distance between the microscope slide and the cover slip. Lastly, the visualization on a microstructure scale of the specimens was evaluated with an inverted Stellaris 5 DMi8 (Leica Microsystems CMS GmbH, Amsterdam, The Netherlands). White Light Lasers (WLL) provided the excitation of Rhodamine B at 543 nm, and a lens of HC PL APO CS2 20x/0.75 DRY was used for taking

Scanning electron microscopy (SEM)
The SEM method as described by Taghian Dinani et al. [39] was followed to obtain information on the microstructure of sheared samples. Frozen samples were cut in a rectangular shape with dimensions of 5 mm × 13 mm. Next, samples were collocated in a closed tube with 10 ml of 2.5% (v/v) glutaraldehyde while lightly shaking for 8 h using a Mini Rocker-Shaker (MR1, Riga, Latvia). The glutaraldehyde was then replaced by demineralized water. The samples were gently rotated overnight using the Mini Rocker-Shaker. Subsequently, the samples were immersed in a series of ethanol at 10, 30, 50, 70, 96, and 100% (v/v) for at least 1 h. Then, the samples were dried using a critical drying point (CPD 300, Leica, Vienna, Austria) and fractured manually perpendicular to the shear-flow direction. Consequently, the samples were fractured by hand parallel to the shear direction. Then, the fractured samples were mounted on the stubs via carbon cement glue and coated with 12 nm of tungsten (SCD 500, Leica, Vienna, Austria). Lastly, the surface of the samples was evaluated at magnifications of 250× (~ 300 μm) and 10,000× (~ 5 μm) by a field emission scanning electron microscope (Magellan 400, FEI, Eindhoven, the Netherlands) with a secondary electron detection of 13 pA and 2.00 kV.

X-ray microtomography (XRT)
To study the distribution of entrapped air, we followed the XRT procedure as described by Schreuders et al. [30]. Frozen sheared products were cut in a rectangular shape with dimensions of 9 mm × 20 mm parallel to the shear-flow direction. Then, the specimen was collocated in an Eppendorf tube to keep moisture content. Next, the sample was placed in an XRT (GE Phoenix v|tome|x m tomographer,General Electric Go., Wunstorf, Germany) with a spatial resolution of 6.0 µm using a distance between object and the X-ray source of 23 mm. An operating voltage of 75 kV, a current of 80 µA, and a power of 6.0 W were used. Images were taken using a GE DXR detector array with 2024 × 2024 pixels. Each specimen was pictured by taking 1501 images over 360° with a 0.24° step ratio and an exposure time of 150 ms when the first picture was omitted. Then, 3D structures were calculated via back projection and the software of Image Reconstruction GE software (Wunstorf, Germany). The 3D images were analyzed by the software of Avizo3D 2021.2 imaging (Thermo Fischer Scientific, Waltham, Massachusetts, USA). Finally, the overall amount of air entrapped in the structure (void fraction) was calculated by dividing the air volume [cm 3 ] by the sample volume [cm 3 ].

Statistics
All the experiments were analyzed with SPSS software (Version 28.0, IBM Corporation, USA) by a general linear model procedure (GLM) which performs regression analysis and analysis of variance (ANOVA) for the dependent variables with a homogeneity test. A significance level of 95% (p ≤ 0.05) was considered for all dependent variables. Consequently, if there was a significant effect, Duncan's post hoc was performed to evaluate the differences among the means of the dependent variables. All the tests except the XRT test were performed at least three times and all the results were shown as the mean ± the standard deviation (SD) in this study. XRT experiment was performed in duplicate for each sample.

Results and discussion
Macrostructure Figure 3 shows  Figure 3 shows that all products have a similarly rounded form except PPI. WG.Ca, which was fragile, presenting some fractures and holes. Moreover, some differences in the color of these products can be seen in this figure wheat gluten, calcium chloride, iota carrageenan, after PPI addition, and before PPI addition, respectively fiber formation. PPI.WG.Ca had a different structure in comparison with other products as it had a fragile and crumbly structure without any visible fibers making it difficult to fold without breaking. This product is not similar to the plantbased meat alternative containing 40 wt % dried ingredients including PPI (19.5 wt%), WG (19.5 wt%), and sodium chloride (1.0 wt%) processed at 120 °C with visible fibers that was reported by Schreuders et al. [30]. The differences could be because of the different formulations (less dried ingredients in our study (35 wt%) and using Ca instead of NaCl) or because of the different processing temperature (100 °C in our study) in the HTSC. Schreuders et al. [30] mentioned that a weak and fragile dough product without any fibers was obtained at 95 °C and small fibers were observed at 110 °C in the PPI-WG blend. Thus, the structure of PPI.WG.Ca processed at 100 °C in our study is in line with the structure of dough obtained at 95 °C and the structure with small fibers obtained at 110 °C reported by these researchers. Figure 4 shows that there were no significant macrostructural differences for the addition of IC without Ca after PPI addition (PPI.WG.IC,A) or before PPI addition at different Ca,A has the best fibrous structure in this figure. It could be suggested that adding IC after PPI in a PPI.WG.Ca mixture promotes protein phase alignment and fiber formation [12]. The results indicate that the hydration of PPI in a Ca solution is important for the PPI solubility and for structure formation in the PPI.WG.IC.Ca,A product. The initial absorption of Ca in the PPI phase could provide the opportunity for the WG to create the gluten network required for fibrous structure formation [3,30,36]. Finally, differences between the PPI.WG.IC.Ca,A sample containing both IC and Ca, compared to PPI.WG.IC,A sample containing IC without Ca could be attributed to the calcium ions, which were earlier found to be essential for forming anisotropic structures in case of caseinate [44,45]. IC has an affinity to calcium ions, forming elastic gels [14]. Another possible explanation could be the effect of Ca on the viscosity of the protein blends. Krintiras et al. [16] mentioned that anisotropic structure formation is enhanced in highly viscous systems. Furthermore, the viscosity of the two phases affects the deformation of the dispersed phase [4,5].

Color
Color is an essential attribute of products that aim at resembling meat, and good control over the color may help the design of plant-based meat alternatives that are more attractive and acceptable to customers [18,19]. Figure 5 Figure 6 summaries the tensile stresses of the different materials; Figure S1. A and B in the Appendix section show the tensile strains and Young's modulus, respectively. In Fig. 6 Fig. 6, the tensile stress in the parallel direction to the shear flow is larger than those in the perpendicular direction, indicating at least some mechanical anisotropy [12]. There are no significant differences (p > 0.05) within the treatments in the PPI.WG.IC group in the addition of IC before or after hydration, mixing time, or mixing temperature for parallel direction. Regarding the perpendicular direction in the PPI.WG.IC group, only PPI.WG.IC,B75°C-15 min is significantly lower than PPI.WG.IC,B25°C-30 min treatments in this group (p ≤ 0.001). Regarding all treatments in the PPI.WG.IC.Ca group (containing both IC with Ca), no significant differences (p > 0.05) in shear stress values in both parallel and perpendicular directions were found. Therefore, the hydration temperature and time for IC are not parameters that can be used to influence the tensile stress. Although it is believed that calcium improves protein-protein interactions ( [44,45], the PPI.WG.IC.Ca treatments with Ca gave lower tensile stress in both parallel and perpendicular directions than PPI.WG.IC treatments. A similar effect was shown in a study on meat, in which a weaker structure was found after addition of Ca [11]. Mcmahon et al. [22] found that a low calcium concentration led to weak bonds among proteins. On the other hand, Pan et al., [26] reported low WHC values and strengths with high concentrations of Ca in mixtures of carrageenan and porcine myosin. The gels became more compact and stronger upon an appropriate concentration of Ca addition. [48,49]. observed that a low concentration of Ca in a mixture of soy and whey protein leads to fewer aggregates in the protein phase and weaker gels. The gelation process could be affected positively by the proper concentration of Ca that has effects on the cross-linking between molecules such as hydrogen bonds and hydrophobic interactions [48,49].

Tensile strength
Taghian Dinani, Broekema, et al., [36] found an increase in the tensile stress when IC is added to a 40 wt% PPI. WG mixture containing 1% Ca and processed in the HTSC (120 °C, 15 min, 30 rpm). Therefore, it could be suggested that adding different concentrations of Ca or using different processing conditions for the preparation of protein blends or for the processing of protein blends in the HTSC explains the reduction of the tensile stress for the treatments within the PPI.WG.IC.Ca group compared to our previous study. In summary, the treatments in the PPI.WG.IC group give higher tensile stress parallel to the shear-flow direction, while PPI.WG.IC,A results in similar tensile stresses for both parallel and perpendicular directions to the shear flow in this group.

Water holding capacity (WHC)
One of the most important attributes of the plant-based meat alternatives is the juiciness. The development of juiciness is often related to the WHC of plant-based meat alternatives [18]. Figure 7 depicts that WHC data range from − 1 ± 3.3% for PPI.WG.Ca to 124.3 ± 19.3% for PPI.WG.IC,B75°C-30 min. Although the WHC of PPI.WG.IC,B75°C-30 min treatment Fig. 6 Tensile stress (kPa) values of the products with different formulations and preparation procedures processed in the HTSC. In this figure, PPI, WG, Ca, IC, A, and B correspond to pea protein isolate, wheat gluten, calcium chloride, iota carrageenan, after PPI addition, and before PPI addition, respectively. Moreover, the first, second and third axis X lines from bottom to top show the formulations, hydration mixing temperatures (25 or 75 °C), and hydration mixing times (15 or 30 min), respectively. Dark red and black letters above and below data points display statistically significant differences in outcomes (p ≤ 0.001) Generally speaking, hydrocolloids can be used as replacers for fat because of their high-water retention and propensity to form soft gels. They increase the viscosity and the plasticity of plant-based meat alternatives [23]. A higher WHC could result from the dry matter holding more water, or less dry matter getting lost from the sample into the water bath [37]. The PPI. WG.Ca exhibited high dry matter loss, and thus even resulted in negative WHC values for some replications. PPI.WG.IC yields statistically higher and different WHC (p ≤ 0.001) than PPI.WG, PPI.WG.Ca, and PPI.WG.IC.Ca treatments. J. Zhang et al. [48,49] and Wang et al. [43] found that a high amount of Ca improves protein-protein interactions and cross-linking. Thus, a lower WHC could be expected for PPI.WG.Ca and PPI.WG.IC.Ca. Finally, insignificant differences (p > 0.05) are found between different mixing times (15 min or 30 min) and temperatures (25 °C and 75 °C) for similar treatments in each PPI.WG.IC and PPI.WG.IC.Ca group.

Confocal laser scanning microscopy (CLSM)
CLSM was performed for the different products described in Table 1 and Sect. "Preparation of different protein blends" at the scale of 100 µm in the shear-flow direction. In Fig. 8, PPI.WG (control) did not show considerable spatial differences in red color intensity. However, adding Ca and IC to the mixtures resulted in much more spatial variation in intensity, indicative of more heterogeneity than PPI.WG. For instance, PPI.WG.Ca contains large and dense agglomerates which are intensely colored. The Ca will result in more cross-linking in WG, and therefore increases the concentration in the WG rich domains. Moreover, as Ca absorbs water by itself, it decreases the availability of water to WG. It is clear from Fig. 8 that the addition of Ca leads to changes in microstructure and promotes the aggregation of spherical particles. For instance, PPI.WG.Ca contained more pseudo spherical structures with high-intensity red color than PPI. WG treatment [48,49]. described that calcium ions promote the formation of small and dense particles, and they found that the addition of more Ca in a Mesona chinensis polysaccharide-whey protein isolate mixture resulted in the formation of large and dense agglomerates.
The addition of IC changed the product microstructures and increased the differences between high and low-intensity red colors in the pictures compared to PPI The spatially different concentrations of PPI and WG causes differences in red color intensity. The lower fluorescence intensity corresponds to the PPI diluted phase and the higher red color intensity indicates the WG rich domains [30]. Less variation in color intensity was found with PPI.WG.IC group than with PPI.WG.IC.Ca. We suggest that the addition of Ca with IC in the formulations results in an IC network that absorbed more water. This will then reduce the water available to be absorbed by WG.
Comparison of PPI.WG.IC and PPI.WG.IC.Ca groups show that when IC is added to the mixture after PPI hydration, it competes with PPI for absorbing water, reducing the differences between PPI and WG. Thus, less differences in red color intensity with the addition of IC to the PPI.WG.IC,A and PPI.WG.IC.Ca,A are seen in comparison with PPI.WG.IC,B and PPI.WG.IC.Ca,B products, respectively. It is interesting also to notice in Fig. 8  WG.IC,B25°C-30 min), respectively. At 75 °C, IC absorbs considerably more water, and, thus, decreases the water absorption by PPI, increasing the homogeneity. In this condition, also the black regions are more in the samples with mixing IC at 75 °C for 15 and 30 min compared to the mentioned samples in PPI.WG.IC and PPI.WG.IC.Ca groups. Black regions could be because of domains with IC hydrocolloid only or elongated air bubbles. An explanation could be that IC absorbs more water and thus, its volume fraction increases and more black color regions in these samples can be observed. Figure 9 shows SEM pictures of the described products (  Fig. 9 The SEM pictures of products with different formulations and preparation procedures processed in the HTSC In this figure, PPI, WG, Ca, IC, A, and B correspond to pea protein isolate, wheat gluten, calcium chloride, iota carrageenan, after PPI addition, and before PPI addition, respectively. Moreover, orange, blue, green, and pink boxes indicate cauliflower-like, sponge-like, spider web, and combined sponge and spider morphologies, respectively. The red and yellow arrows indicate string and glubules, respectively XRT images of solid part (in the purple rectangular shape) and air bubble distribution (in pink color) of the products with different formulations and preparation procedures processed in the HTSC. In this figure, PPI, WG, Ca, IC, A, and B correspond to pea protein isolate, wheat gluten, calcium chloride, iota carrageenan, after PPI addition, and before PPI addition, respectively structure was noticed in PPI.WG.IC.Ca,B25°C-30 min. Similarly, the spider web morphology is more pronounced in PPI.WG.IC.Ca,B75°C-15 min than in PPI.WG.IC. Ca,B75°C-30 min. Therefore, it seems that increasing the mixing time in the PPI.WG.IC.Ca products resulted in a more compact structure.

X-ray microtomography (XRT)
The XRT images in Fig. 10 show the bubble counts and overall air fractions in the processed products (Fig. 11A (Fig. 11A, the difference in the total air fraction is much lower; indicating that these materials have on average much larger bubbles or cavities. (Fig. 11B). Air bubbles have been found to be necessary for producing a more pronounced fibrous structure in case of SPI-based products [4,5]. Mentioned that anisotropy appears when the deformation of phases occurs, and air bubbles are a separate phase, but Schreuders et al. [30] indicated that the hypothesis of air-enhancing anisotropic structure is not in line with the fiber formation in PPI.WG mixtures. In our study also the sample with the most pronounced fiber structure (PPI. WG.IC.Ca,A in Fig. 4) does not possess the highest bubble numbers and air fraction (Fig. 11), so we agree with Schreuders et al. [30].