3.1. Proximate Compositions of TVP
Proximate compositions, including protein, moisture, ash, fat and total carbohydrate contents of TVPs, are presented in
Table 1. As shown, TVP samples varied significantly in protein content among the diverse protein sources, with textured pea proteins overall having the highest protein amount (samples 15–21, 62.4 to 76.6%), which was closely followed by textured wheat gluten (samples 22–25, 64.4 to 72.1%) and textured mixed proteins (samples 27–28, 66.3 to 68.3%). Textured soy proteins (samples 1–14, 50.0 to 55.8%) and the textured chickpea protein (50.4%) were the lowest in protein content. Protein is the most paramount component of TVP. A protein content of 50–70% is generally required to form fibrous structures during extrusion [
21]. In addition, soy, in comparison with other proteins, such as pea protein, is relatively easier to texturize when forming fibrous structures at lower protein content, as evidenced by the fact that many soy-based TVPs are made from protein concentrates, while pea TVPs are derived from protein isolates [
22,
23,
24]. Overall, the wide range of protein concentration (50.0 to 76.6%) in the studied samples enabled the formation of fibrous textures.
A similar tendency was also observed in fat content. TVPs derived from pea proteins exhibited a substantially higher fat content (in an average of 6.0%) when compared with textured soy proteins (in an average of 2.7%) or wheat gluten (in an average of 2.8%). The textured mixed proteins located in a high range of fat content (samples 27–28, 6.0 to 6.6%) as well, while the textured chickpea protein (2.0%) was in the lowest range. On the contrary, textured soy proteins registered the highest ash content (5.6 to 7.1%), followed by the descending order of textured pea proteins (3.8 to 5.6%), textured mixed proteins (4.8 to 4.9%), textured chickpea protein (4.6%) and textured wheat gluten (2.4 to 3.0%). The higher ash content possibly arose from a higher amount of minerals in the raw materials prior to texturization. The moisture content of TVPs differed significantly from 4.8 to 8.5%, although with no specific tendency observed among the various protein sources, which might result from the differences in the extrusion conditions and the post-drying processes. The total carbohydrate content was found highly oppositely correlating with protein content (r = −0.984,
p < 0.01,
Table 2. The textured soy proteins (27.4–35.4%) and textured chickpea protein (34.9%) exerted the highest total carbohydrate content. TVPs sourced from wheat gluten (14.7–22.0%) and mixed proteins (14.8–18.2%) contained considerably lower amounts of carbohydrate, whereas textured pea proteins were observed in the lowest place (4.8–20.9%). The variations in chemical compositions of the TVPs are likely responsible for the differences in the physicochemical and textural properties of TVPs before and after formulating to patties.
3.2. Physicochemical Characteristics of TVP
Protein solubility commonly functions as a vital indicator of the degree of protein texturization [
11]. Upon extrusion cooking, the protein is thermally denatured, with a series of unfolding and aggregation, leading to a decrease in soluble protein. Thus, lower solubility of textured proteins is usually observed compared with their native counterparts [
11,
20]. The soluble protein content of the studied TVPs ranged significantly between 43.0 and 90.3%, as shown in
Table 3. An ascending trend was observed as follows: wheat gluten-based TVPs (samples 22–25, 43.0 to 48.5%) < pea-based TVPs (samples 15–21, 59.7 to 73.5%) < soy-based TVPs (samples 1–14, 74.9 to 90.3%). Meanwhile, the chickpea-based TVP (sample 9, 79.5%) exerted comparable solubility to soy-based TVPs, while the protein solubility of pea/chickpea- (sample 27, 68.6%) and pea/navy bean- (sample 28, 67.1%) mixed protein based TVPs fell within the range of pea-based TVPs. The differences of solubility among the various protein sources may arise out of their intrinsically different molecular structures, as well as varying degrees of protein denaturation during extrusions with diverse conditions.
A significantly negative relationship existed between the protein solubility and the protein content (r = −0.775,
p < 0.01), as presented in
Table 2. Indeed, a higher protein content could possibly contribute to a greater extent of protein denaturation during extrusion cooking, which resulted in an increase in protein texturization and insoluble proteins, thus lowering the solubility [
11,
18,
20]. Moreover, the intermolecular disulfide bond was suggested as the major force being responsible for the fiber formation of TVP [
25,
26]. In contrast to legume proteins, wheat gluten contains relatively higher levels of methionine and cysteine [
11]. Such sulfur-containing amino acid residues are likely to result in more disulfide cross linkages during texturization, which thereby lead to an increment of molecular weight and the insolubility of proteins [
27]. This could possibly explain the lowest protein solubility of the textured wheat gluten samples (in an average of 45.8%) in the current study. However, the structures of extrudates are complex and are usually stabilized by the collective contributions of hydrophobic interactions, hydrogen bonds, disulfide bonds and their interactions [
28]. Studies also showed that the importance of non-covalent bonds outweighed covalent bonds [
28]. Overall, a lower protein solubility after extrusion is usually concluded as a greater protein denaturation and texturization.
WAC or OAC indicates the ability of a sample to absorb water or oil at the macromolecular level. The amphiphilicity of a protein enables its ability to interact with both water and oil [
29]. As such, WAC and OAC are reliant on the availability of polar and non-polar amino acid residues, as well as the protein’s micro- and macro-structures [
29]. A lower presence of hydrophilic and polar amino acids over the surface of the protein molecule contributes to lower WAC, while higher availability of hydrophobic residues is responsible for higher OAC.
Table 3 shows the WAC of TVPs varying from 1.5 to 2.9 g/g, being independent of protein types or protein contents but potentially associated with the available amounts of polar amino acids in each sample. Meanwhile, an improved entrapment of water has been reported as a consequence of the formation of a protein matrix that is induced by protein denaturation during extrusion [
24]. In this study, the wheat-gluten-based TVPs may take great advantages of this phenomenon, as wheat gluten exerted statistically lower protein content but exhibited comparable WAC to that of pea-based TVPs (in an average of 2.1 and 1.9 g/g, respectively). Apart from proteins, the higher carbohydrate contents in the current extrudate samples may also play an important role in the WAC results, since more starch granules were able to absorb more water after gelatinization [
9], which might account for the similar WAC of textured soy proteins (in an average of 2.1 g/g) to that of textured pea proteins, although the former were significantly low in protein content (
Table 1).
It is worth noting that OAC was substantially greater for TVPs derived from pea proteins (samples 15–21, 0.82 to 1.04 g/g) than those made with wheat gluten (samples 22–25, 0.75 to 0.86 g/g) or soy proteins (samples 1–14, 0.69 to 0.84), which occurred possibly due to a higher content of hydrophobic amino acids in pea proteins (30.26 g/100 g protein) than in others (28.23 g/100 g protein for wheat gluten and 26.21 g/100 g protein for soy protein), as confirmed by Samard and Ryu [
11]. Moreover, OAC was found to positively correlate with fat content (r = 0.852,
p < 0.01) and protein content (r = 0.711,
p < 0.01) of TVPs (
Table 2). Joshi et al. [
30] found that full-fat oilseed flours exhibited lower OAC than their defatted counterparts, as the removal of the fat greatly improved the protein proportion, thus allowing better capillary attraction between the protein and the oil [
31]. However, a relatively higher fat content, which was not able to significantly lower the protein content, favored the OAC results in the current study, as the non-polar lipid may enhance the interactions with oil on the basis that protein was the predominant composition governing the OAC of the studied TVPs. On the other hand, WAC and OAC may associate with the extent of denaturation, as extrusion cooking results in the unfolding of proteins and the exposure of more hydrophobic sites [
24]. Thus, increasing the protein concentrations may not only contribute to a higher amount of hydrophobic amino acids but is also potentially responsible for the greater extent of protein denaturation induced by extrusion, thereby introducing more available hydrophobic sites, which contribute to greater OAC values. Osen et al. [
24] reported that extrusion heat treatment enhanced the OAC of pea protein isolate due to the exposure of more hydrophobic sites. Meanwhile, the polar carbohydrates may, on the other hand, have a negative effect on the extent of interactions with oil, as shown an opposite relationship between carbohydrate content and OAC (r = −0.763,
p < 0.01,
Table 2). In summary, WAC and OAC are multifactor dependent, including protein composition, protein denaturation, as well as the extent of interactions with water and oil [
24].
3.3. RVA Pasting Properties of TVP
Viscosity plays a crucial role in altering the flow behavior and the mechanical energy input in extrusion cooking [
32]. In this study, RVA pasting profiles were obtained to understand the viscosity properties of proteins after texturization. As shown in
Table 3, the TVPs behaved dramatically differently upon hydration, heating and cooling under a slow shear. During heating, all samples, regardless of the protein types, endured vast elevation in their viscosities, achieving significantly different peak viscosities ranging from 502 to 4252 cP. However, the peak viscosities were diminished to some extent from the shear in the case of textured wheat gluten (samples 22–25), as indicated by the lower final viscosities compared with their corresponding peak viscosities. Differing from this, the TVPs derived from other sources were increasing in viscosity throughout the holding and cooling, implying their better abilities against shear thinning, while forming viscous pastes or gels upon cooling, which may benefit the texture of the final products. The reduction in the final viscosity could possibly be related to the low protein solubility of textured wheat gluten (
Table 3) on the basis of understanding that lower protein solubility is indicative of a more complete texturization, thus a higher denaturation degree, as stated earlier, and the already denatured proteins may have induced weaker protein–protein interactions upon heating, which weakened the resistance to shearing and thereby decreased the final viscosities.
Despite distinct variations in viscosities, both peak viscosity and final viscosity were found positively correlating with WAC (r = 0.621 and 0.549, respectively,
p < 0.01,
Table 2). This finding is in line with previous studies, where a protein with higher WAC was able to absorb more water, which resulted in higher viscosities [
24]. On the other hand, in contrast with pea or wheat gluten, textured soy proteins (samples 1–14) generally required a longer time (ranging from 6.6 to 7.0 min) to achieve peak viscosity, indicating that soy proteins need more time to hydrate and bind water and higher temperatures to denature before reaching the maximum viscosities. This result may be attributed to the relatively higher carbohydrate amount in such samples (
Table 1), which may interfere with the hydration and swelling process of proteins, thus retarding the denature time (r = 0.633 between carbohydrate and peak time,
p < 0.01,
Table 2).
3.5. Rehydration Property
Water is critical in meat products to endow the appropriate texture and juiciness, so as to ensure customer acceptability. RHC, referring to the amount of water that could be held by the intact TVP upon rehydration, is an imperative factor affecting the meat-like texture of plant-based meat analogs [
9]. In the current study, the RHC values of all samples were significantly different to each other, from 1.5 to 4.2 g/g, as demonstrated in
Table 4. Differences in RHC are dependent on protein types, interactions between protein-water molecules, and water–water molecules [
11,
20] but are more closely related to the product structure, in particular, the porosity and air cell size [
9,
20]. Here, the external appearance and internal structure of TVPs after hydration are distinguished in
Figure 1 and
Figure 2. As shown, the TVP samples showed porous structures with various sizes and numbers of air cells, which may have resulted from the different degrees of expansion during extrusion. It is worth mentioning that the images were taken as their naturally displayed directions (longitudinal or horizontal cross sections of extrusion), since the current samples were commercially obtained and were difficult to cut purposely due to the limitation of their shape and size. This could explain why some TVPs exhibited more elongated cells, while others had pores with smaller diameters (
Figure 2). Diverging from some previous studies that related a higher RHC to a lower bulk density, as products with low bulk density may possess higher porosity, which allows for faster water uptake and consequently leads to a better water-holding capacity [
9,
11], no such clear correlation occurred among the current samples. More compact products, such as samples 8, 14, 17, 21, 22, were also able to retain a great amount of water, as evidenced by the relatively high RHC values (3.3 to 3.8 g/g), while lower RHC also occurred in more porous and fibrous structures (samples 1, 2, 6, 10, 13, 16, 24, from 1.5 to 2.5 g/g). The inconsistence may be due to the difference in determining the RHC. The comparatively longer draining time in the current study (1 h) may permit more water to drain off from the more porous protein network, as a higher number of air cells is likely to result in easier water release caused by the gravitational force, whereas a shorter draining time possibly only allows water to drip and evaporate from the surface. In addition to the pore number, the size of the air space is also important to retain water [
32].
3.6. Textural Properties of TVP
Texture is undoubtedly the most crucial attribute characterizing the quality of textured plant proteins, since a desirable texture that mimics the real meat is the main task of meat analogs.
Table 4 shows the textural properties of hydrated TVPs in terms of hardness, resilience, cohesiveness, springiness and chewiness. Hardness is the maximum force required to attain a defined deformation [
34]. It differed in a wide range, varying from 400 to 2428 g, among the studied samples (
Table 4). Hardness may be indicative of the degree of protein texturization [
20]. In this sense, a higher presence of protein content in the starting material is assumed to increase the degree of texturization and protein cross-linking, which prevents further expansion and leads to a higher hardness [
20]. Webb et al. found that hardness decreased with the increasing inclusion of chickpea flour, from 10% to 30%, which interfered with the protein–protein interactions [
9]. In addition, the hardness and RHC of TVPs appeared to be negatively correlated (r = −0.765,
p < 0.01,
Table 2), agreeing with some previous studies [
9,
26] that extensive hydration of TVP usually leads to a softer texture [
35,
36]. Additionally, the diversity of hardness may arise from the various processing variables. Rising barrel temperature and lowering feed moisture have been reported to associate with higher hardness [
37]. Overall, it is rather difficult to manifest a clear clue addressing the wide range of hardness here, since all the studied samples came from different commercial sources and were made under diverse extrusion conditions.
Resilience measures how a sample recovers from deformation with regard to speed and forces. As shown in
Table 4, resilience values extended from 16.4 to 37.5%, displaying no specific tendency among the protein sources, although being inversely correlated with bulk density (r = −0.665,
p < 0.01,
Table 2). Products with higher bulk density potentially possess more compact structures, which likely impair the resilience. Here, TVP samples exhibited relatively high springiness, going from 79.1 to 100.5%, suggesting good abilities of TVPs to regain their original form after compression. Likewise, springiness was negatively related to bulk density, with r = −0.724 (
p < 0.01,
Table 2). The lower bulk density benefits a higher porosity and loose structure, thereby enhancing the springiness. Cohesiveness indicates the strength of internal bonds and inter- and intra-actions constituting the product [
34]. Samples exhibited a cohesiveness of 0.53 to 0.72 in the current study, which might be a response to the different degree of interactions formed during texturization and rehydration [
12]. In addition, chewiness represents the energy necessary to masticate a solid product for swallowing [
34]. As expected, the wide spectrum of chewiness (276 to 1530 g) positively corresponded with hardness (r = 0.977,
p < 0.01). The lower chewiness may largely be a result of a higher RHC, which leads to a softer texture (r = −0.737 between chewiness and RHC,
p < 0.01,
Table 2).
3.7. Cooking Properties of TVP-Based Patties
The visible appearance of TVP-based patties before and after cooking is presented in
Figure 3. It is worth mentioning that the patties in this study had the same formulation. In addition to the different types of TVPs, all the other ingredients (salt, pigment, binder, etc.) were added in the same amounts. Thus, the diverse properties of patties were assumed to result from the various properties of the TVPs. The effect of cooking on patties was investigated by measuring cooking loss, diameter shrinkage, moisture retention and fat retention. Cooking loss is an important parameter evaluating the textural and sensorial attributes of meat products with regard to juiciness, tenderness and also the yield of the final product [
35]. It is mainly caused by the loss of liquid (moisture and fat) during the cooking process [
38] and is linked to different variables, such as cooking time, temperature and method, type and amount of particular ingredients in the formulation [
39,
40].
The cooking loss of TVP-based patties ranged vastly from 11.6 to 18.5% (
Table 5), irrespective of protein types. A positive relationship was observed between the RHC of TVPs and the cooking loss, as stated in
Table 2 (r = 0.679,
p < 0.01). At higher RHC, a relatively higher amount of water was introduced to the meatless patty, causing the proportional decrease in solid content on the basis that the same total amount of hydrated TVP was incorporated. Upon heating, the hydrophobic residues in the proteins became exposed; the heated TVP consequently contributed less hydrophilic interactions with water, which resulted in a leakage of water, and thus, a high cooking loss [
35]. On the other hand, the methylcellulose in the formulation served as a binder that created a network upon protein hydration and helped combining the ingredients together [
7]. It is supposed that the cage-like water molecules encircle the hydrophobic methyl residues of the methylcellulose polymer. Nevertheless, the increasing temperature disrupts the cage structure, causing the release of water [
41]. In light of this, a higher cooking loss is likely to occur in patties formed by TVPs with higher RHC. This finding is in accordance with many other studies. Wi et al. [
35] found a typical increase in cooking loss from 12.5 to 14.5% as the amount of water increased in meat analogs. The same trend was also reported by Sakai et al. [
41], where the increasing amount of added water elevated the cooking loss.
It is also interesting to note that patty cooking loss was positively associated with protein viscosities (r = 0.605 and 0.660 for peak and final viscosity, respectively,
p < 0.01,
Table 2). In this case, it might be hypothesized that the enhanced hydrophobic interactions induced by protein denaturation upon heating helped form a tighter network, which not only increased the viscosity but also decreased the free space within the protein matrix, thus reducing water penetration and uptake and increasing the cooking loss.
Cooking causes meat shrinkage due to protein denaturation, change of structure, moisture loss and fat drainage [
42]. As expected, the reduction in patty diameter was highly correlated with cooking loss (r = 0.786,
p < 0.01,
Table 2), together with a positive correlation with RHC (r = 0.679,
p < 0.01,
Table 2), which ranged from 4.4 to 9.5% (
Table 5). This degree of shrinkage fell within the spectrum of 3.6–12.3% for commercial textured vegetable protein (C-TVP) and textured isolate soy protein (T-ISP) based patties, as reported by Bakhsh and others [
43].
Proteins form a gel matrix during the cooking treatment, which is able to retain the essential components [
42]. Moisture and fat retentions refer to the capabilities of a product to retain water and fat after cooking. They are crucial factors ensuring the sensory quality and acceptability of meat products.
Table 5 displays the moisture retention of TVP-based patties varying from 73.2 to 80.5%, while the fat retention differs from 74.3 to 92.4%, being unaffected by protein sources. The diversity of these parameters was possibly derived from the different degrees of protein denaturation and the extent of the interactions between water/oil and the TVP structure [
18]. Both moisture retention and fat retention were inversely related to cooking loss (r = −0.655 and r = −0.684, respectively,
p < 0.01,
Table 2), as a higher cooking loss usually occurs when a patty loses more fat or moisture [
38,
42]. In addition, the negative correlation between moisture retention and protein viscosity (r = −0.530,
p < 0.01,
Table 2) may again give an insight into the enhancement of the hydrophobic bindings, which allowed rising viscosity and retaining less moisture. Meanwhile, less fat was likely to be held due to less free space and enhanced rigidity of the protein gel, which may help explain the negative relationship between fat retention and pasting viscosity (r = −0.601 and r = −0.552 for peak and final viscosity, respectively,
p < 0.01,
Table 2). However, fat retention is a complex parameter, which may be associated with several other chemical and physical mechanisms [
38].
3.8. Textural Properties of TVP-Based Patties
Table 6 shows the textural properties of cooked patties derived from different TVPs. While hardness in the patty form was highly related to that in the hydrated counterparts (r = 0.885,
p < 0.01,
Table 2), the former was generally greater than the latter (559 to 2767 g vs. 400 to 2427 g), which was possibly due to the methylcellulose binding during the patty formation and gelling during cooking that resulted in the compacting of the material. During the cooking process, methylcellulose gradually loses its hydrated water and is likely to bind together owing to the extensive hydrophobic interactions, which highly favors the thermal formation of gels [
43]. The strong gels thereby toughen the texture of the final product. Consistent with TVP hardness, the hardness in cooked patties varied negatively with the RHC of TVPs (r = −0.791,
p < 0.01,
Table 2), since a higher water content commonly forms more softened meat analogs [
35,
36]. Meanwhile, when TVPs with higher RHC were incorporated, a relatively lower solid content was induced to the patty. The decrease in the solid amount may have caused the reduction in hardness as well. However, disagreeing with some previous studies [
44], the hardness in the current patties was inversely associated with cooking loss (r = −0.618,
p < 0.01,
Table 2). It is possible that TVPs with a high RHC, although undergoing a higher cooking loss, as previously stated, may have still retained a relatively higher amount of water, and the softening effect caused by the residual water played a more important role than the toughening impact induced by the shrinkage, which thereby resulted in a lower hardness in such samples compared with those with a lower RHC but also lower cooking loss.
A moderately negative correlation existed between patty hardness and peak viscosity (r = −0.599,
p < 0.01,
Table 2). Given the above explanation, the comparatively higher water remainder in samples with high RHC may not only result in a tender texture of a patty, but also contribute to a relatively higher viscosity of the protein due to a higher retention of water during cooking. It is also worth noting that higher hardness was related to an increase in fat retention (r = 0.537,
p < 0.01,
Table 2). Barbut and Marangoni reported that oil droplets could help connect the protein–protein interactions due to their smaller size but larger surface area [
45]. Therefore, an increasing oil globule in products with higher fat retention incremented such hydrophobic linkage and formed a more compact and firmer gel network among the protein matrix, thus enhancing the resistance to compression.
Differing from hardness, other textural attributes were all found to reduce in the patty form in contrast with the hydrated TVPs before binding (
Table 4 and
Table 6). The resilience of the cooked patties remained with substantially lower values, going from 4.1 to 11.5% (
Table 6). The observed lower results in patties made from textured soy proteins could be attributed to the better ability of soy protein to form strong structures, thus a more compact texture and higher bulk density (r = −0.506 between resilience and bulk density,
p < 0.01,
Table 2). Similarly, a dramatic decrease in springiness was observed, as most values ranged from 50 to 80% (
Table 6), implying that they were more prone to be deformed in the patty form. Apart from a more compacted form induced by methylcellulose binding, the fat content introduced in the formulation that helped fill the interspace within the protein matrix may have also resulted in a reduction in springiness. Cohesiveness was similar to the above, in that there was a decline from the hydrated extrudates to the patty form (0.53–0.72 vs. 0.19–0.38). Cohesiveness is related to intermolecular attractions, which are able to hold the elements together [
44]. In a food product, cohesiveness also represents the extent to which the food can be deformed before it ruptures [
44]. Here, it may be more useful to regard the cohesiveness of the cooked patties as the strength to withstand fracture in a patty as an entirety rather than to disintegrate the TVP particles, which resulted in the difference before and after the formation of patties. As for chewiness, significantly lower values were found in the patty form (93 to 391 g) compared with those of hydrated TVPs before binding (208 to 1530 g). The lower force required to chew the cooked patties was possibly due to the protein denaturation caused by the cooking treatment, which altered the protein conformation and structure.
Shear force represents the maximal force needed to cut a patty, which can be interpreted as an indirect measurement of product tenderness [
5]. Here, the shear forces ranged from 69 to 527 g among the studied patties. As reported elsewhere [
43], shear force behaved in a significantly similar manner to hardness (r = 0.778,
p < 0.01,
Table 2), with RHC being the predominant affecting factor in the current study (r = −0.621,
p < 0.01,
Table 2). In this respect, a higher RHC of the TVP would be a favorable implication, achieving lower hardness and shear force, thus a softer and more tender texture.
Compressed juiciness refers to the percentage weight loss of cooked patties established in a compression test. As found in
Table 6, juiciness in cooked patties varied significantly, from 4.0 to 10.3%. In general, TVPs with higher RHC yielded more juices when formulating a patty, as evidenced by a significantly positive correlation between the RHC and juiciness (r = 0.812,
p < 0.01,
Table 2). This phenomenon was inevitable due to a relatively higher amount of water left within the protein matrix, which was able to be squeezed out. Moreover, the compressed juiciness was negatively correlated to hardness, chewiness and shear force of patties (r = −0.883, −0.540 and −0.653, respectively,
p < 0.01,
Table 2), since a firmer structure was more capable of retaining fluid and more resistant to compression, thus imparting less juice [
46]. Overall, the physicochemical and functional properties of the proteins, the ingredients in patty formulation, as well as the cooking process all play important roles in carrying over the TVP properties into the textures of final products.