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Zhao, D.;  Huang, L.;  Li, H.;  Ren, Y.;  Cao, J.;  Zhang, T.;  Liu, X. Factors Affecting the Structural Quality of Tissue Protein. Encyclopedia. Available online: https://encyclopedia.pub/entry/25974 (accessed on 20 August 2024).
Zhao D,  Huang L,  Li H,  Ren Y,  Cao J,  Zhang T, et al. Factors Affecting the Structural Quality of Tissue Protein. Encyclopedia. Available at: https://encyclopedia.pub/entry/25974. Accessed August 20, 2024.
Zhao, Di, Lu Huang, He Li, Yuqing Ren, Jinnuo Cao, Tianyu Zhang, Xinqi Liu. "Factors Affecting the Structural Quality of Tissue Protein" Encyclopedia, https://encyclopedia.pub/entry/25974 (accessed August 20, 2024).
Zhao, D.,  Huang, L.,  Li, H.,  Ren, Y.,  Cao, J.,  Zhang, T., & Liu, X. (2022, August 09). Factors Affecting the Structural Quality of Tissue Protein. In Encyclopedia. https://encyclopedia.pub/entry/25974
Zhao, Di, et al. "Factors Affecting the Structural Quality of Tissue Protein." Encyclopedia. Web. 09 August, 2022.
Factors Affecting the Structural Quality of Tissue Protein
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Tissue proteins or textured vegetable proteins (with a lean fiber-like structure and chewiness) form the main skeletal structure of plant-based meat alternative products. These can be obtained through techniques such as extrusion, shearing, spinning, freezing structuring and three-dimensional (3D) printing. The main constituents of tissue proteins are proteins, lipids, water, carbohydrates, flavoring agents and coloring agents. The choice of plant protein raw materials, with their various processing characteristics, prior to processing is a decisive factor in structural quality differentiation.

recombinant plant-based meat alternatives tissue protein simulated fat structure optimization

1. Effects of Protein on the Structural Quality of Tissue Protein

Protein is the most important factor impacting the structural quality of tissue protein, as it is the main component of the fibrous structure that mimics meat products. Protein melting and denaturation are prerequisites for the formation of histochemistry. During processing, the combined actions of temperature, shear, pressure and water melt the plant proteins completely, with the molecular chains unfolding in the flow direction, exposing the sulfhydryl groups and creating new disulfide bonds through oxidation. In this way, the protein denatures, losing its original structure, and depolymerizes from the natural spherical aggregated state to form an anisotropic structure. This molten state includes the action of molecules with other substances, in which the protein molecules are rearranged. After sufficient homogenization and oriented arrangement, the mixture is cooled down and characterized, with the protein having been converted into a tissue protein with a structure similar to that of animal meat fibers [1]. Thus, to facilitate this process, it is essential that the proteins used as raw materials contain sufficient sulfur-containing amino acids in their globulin molecular chains [2]. In the next section, the differences and advantages of various raw protein materials, as well as their impact on the structural quality of tissue protein, will be collated and compared.
Table 1, below, presents a comparison of the nutritional properties, processing characteristics and respective limiting factors in the production of different types of raw protein. The practical applications of these products are also summarized to provide a foundation of reference for the selection of raw protein materials for recombinant plant-based meat alternatives.
Table 1. Comparison of the properties of different protein raw materials.

1.1. Legume Protein

Legumes are currently the most important source of protein in the production and processing of tissue protein. Soybeans and other legumes differ in their main components, nutritional values and processing characteristics. For example, soybean is extremely low in carbohydrates, high in fat and protein, and has a more balanced amino acid ratio. Protein content in pea is slightly lower than soybean, but it is one of the major plant protein resources due to the high yield. This section summarizes and analyzes the potential of legume proteins to be used in the production of tissue proteins, using two different categories of legumes as examples.
Soybean protein is a common, high-quality plant protein that can readily replace animal protein [19]. The origin of tofu, which is made from coagulated soy milk, can be traced all the way back to A.D. 965 [20]. Due to the wide availability of high-quality raw soybean materials, the relatively established extraction method and the excellent processing characteristics of anisotropic fiber structures, they are currently widely utilized in the production of tissue protein for recombinant plant-based meat alternatives. The globulin molecular chain in soybean protein contains abundant sulfur-containing amino acids for the manufacturing of tissue protein, which tend to open the chain when subjected to thermal shear, thereby exposing molecular binding sites and contributing to further oxidation to form disulfide bonds and promote the formation of fibrous meshwork. The main forces at work in this organizing process are disulfide bonds, hydrogen bonds, hydrophobic forces and electrostatic interactions, with various proportions of each bond resulting in varied levels of thermal reversibility and structural stiffness [21]. The complex changes in production involve changes in both covalent and noncovalent interactions, with disulfide bonds playing a more important role in the formation of rigid structures and fiber textures than noncovalent interactions [22].
Among legume proteins, pea protein is another of the main raw materials occupying an important position in the plant-based meat alternatives market. This is due to its characteristics of being a non-genetically modified organism (GMO), non-allergenic and estrogen free, with high bioavailability, local cultivation, high yield and low cost [2]. Peas also have considerable development potential due to their processing characteristics and functional food advantages, yet their commercial utilization is currently limited, mainly due to their less-ideal organoleptic properties. However, the processing properties of pea protein can be effectively improved through protein modification. According to a study for the sequential enzyme modification of pea protein, the covalent linking of the protein to hydrophilic polysaccharides can significantly improve the solubility, emulsification properties, water and oil holding capacity of pea protein to form a strong, fibrous and tough tissue protein [23][24]. In another study by Fang et al. [25], pea proteins treated with glutaminase showed higher flexibility, homogeneity and dispersion, and reduced the bean odor and lumpy flocculation in tissue proteins compared to untreated pea proteins. Sandoval et al. [26], who thoroughly investigated the interactions of pea proteins and the mechanism of fibrous structure formation during their processing, concluded that the protein macromolecular network was formed mainly through disulfide bonds during extrusion and cooling, and the formation of the fibrous structure was further facilitated by spinodal phase separation. Thus, protein modification offers a promising approach to enhance the suitability of pea-protein processing, and the obtained modified pea protein has excellent potential in protein tissue processing.

1.2. Grain Protein

The protein content of grains is lower than that of legumes, with poor digestibility and unsatisfactory amino acid composition, particularly in the lack of lysine and threonine [27]. Indeed, the intake of a single category of protein often does not meet the full nutritional needs of the body. For example, most legume proteins are low in methionine and cysteine, while grain proteins are often low in lysine. Consequently, grain proteins require supplementation to improve their biological value in plant-based meat alternatives and to balance the ratio of amino acids in the final product.
Following hydration, wheat protein forms a 3D protein network structure maintained by disulfide bonds, which plays an important role in simulating the fibrous structure of meat, increasing the viscoelasticity and hardness of the product, and improving its color stability, juiciness and water retention [10]. In one study, for example, the extrudate obtained by adding 30% wheat gluten protein under high moisture extrusion conditions was found to have improved organization, firmness and chewiness [28]. The texture was closer to that of real meat, and the viscoelasticity, formability and film-forming properties were enhanced. Guo et al. [29] demonstrated that the proportion of α-helices and reverse parallel β-folds (associated with hydrogen bonding and representing stable secondary structures of proteins) decreased, while the proportion of loose β-turned and randomly curled structures increased in tissue, resulting in improved intramolecular hydrogen-bonding interactions, while promoting water and the retention of flavor substances. Wheat protein plays an important role in increasing disulfide bonding and promoting the formation of fibrous structures because hydrogen bonding is the main interaction force between forming and stabilizing proteins, and disulfide bonding is the key force in forming the fibrous structure of high-moisture extruded tissue proteins [28]. Consequently, wheat protein (with its good processing characteristics) is a more commonly used grain protein, which can be added to plant-based meat alternatives as a second composite protein raw material with a complementary amino acid balance, and has an optimal effect on its histochemical structure quality, which is conducive to the development of high-quality plant-based meat alternative products.

1.3. Non-Plant Classified Protein

Although fungi are not essentially plants, meat analogs made from fungal proteins are often defined as plant-based meat alternatives. Fusarium, Agaricus bisporus and other fungal proteins are sources of high-quality plant-based meat alternatives [30]. Mycelium has a fibrous-like microstructure, and the fungal protein developed from mycelium is similar to that of meat protein, with excellent hardness, elasticity, chewiness and outstanding freshness [31].
Algae proteins, such as chlorella primordialis and spirulina, have also been tested in the manufacturing of plant-based meat alternatives to improve the product quality [32]. Microalgae proteins have good gelation, water absorption, fat absorption, emulsification and foaming abilities, all of which are beneficial in their application in tissue protein production. For example, the addition of spirulina protein in the production of tissue protein has resulted in products with good elasticity, flexibility and fiber properties [17]. Caporgno et al. [33] combined microalgae (30%) and soybean protein tissue to improve the nutritional value, appearance and color properties of the latter and obtain a good tissue protein with significant juiciness. Moreover, compared to other plant protein sources, microalgae have higher growth rates and can adapt to a wide range of growth conditions, making them an efficient source of material for protein production.

2. Effects of Lipids on the Structural Quality of Tissue Protein

During processing, lipids can impact the structural quality and sensory properties of textured proteins. Lipids can act as plasticizers or lubricants during the tissue protein molding process, which can reduce particle interactions in the material and lessen the friction between processing machine and material, resulting in improved texture, viscosity and integrity in the tissue protein product. The combination of different lipid concentrations and processing conditions can produce tissue proteins with differentiated structural characteristics to meet the specific needs of a variety of products [20].
The addition of lipids can also improve the organization of the legume tissue protein, resulting in a product with good fibrous structure and suitable hardness and flexibility. Jia et al. [34] found that the addition of 8% mixed plant oil could affect the protein interactions during wheat gluten protein extrusion by enhancing the polymerization of disulfide bonds, thereby improving the fibrous strength of the tissue protein and obtaining a well-organized product with improved continuity. However, it has been suggested that lipids can adversely affect the anisotropic fibrous structure of organized protein, and that excess lipid can inhibit the formation of fibrous structures, thus reducing the tensile strength and structural quality of tissue protein [35]. Kendler et al. investigated the effects of different lipid contents on the anisotropic fibril structure of tissue protein obtained by processing wheat gluten protein as a base material. No significant differences were observed between the 0% and 2% lipid content samples; however, the anisotropic fibril structure was reduced in the 4% lipid content samples [36]. This was due to the fact that as the lipid content increases, oil droplets aggregate and increase in size, interfering with the polymerization of gluten proteins and leading to a weakened protein network structure.
In general, the addition of lipids has varying effects on process kinetics. Further investigations are required to provide insight into the effects of lipids on processing conditions, protein polymerization, matrix rheological properties and their microstructure, as well as to clarify optimal quantities of lipid additions for various target products. The appropriate proportion of fats and oils can effectively improve fibrous strength and structural quality in the manufacture of organized products with different processing characteristics.

3. Effects of Moisture on the Structural Quality of Tissue Protein

Moisture affects both the fibrous structure and texture of tissue protein, contributing to the unfolding and arrangement of protein molecules during processing and promoting the formation of fibrous structures. It has previously been found that moisture content is a crucial factor affecting the formation of fibrous structure via the extrusion of lupin protein [37]. When moisture content is less than 40%, the hydration of lupin protein is incomplete, with ineffective cross-linking, and the resulting tissue protein fibers are poorly structured and prone to breaking [29]. In high moisture processing conditions, protein molecule aggregation relies mainly on hydrophobic interactions, while disulfide bonds replace hydrophobic interactions as the stabilizing force for protein molecule aggregation in low moisture-processing conditions [38]. During the increase in moisture content from 20% to 60%, disulfide bonding and hydrophobic interaction were found to occur synergistically, thereby facilitating increased fibrillation [39]. It has been suggested that the effect of moisture content on the hardness of tissue protein is most significant during extrusion [40]. Increased free moisture content has a lubricating effect on processing, promotes the fluidity of the material, lowers the processing intensity and pressure, reduces the expansion phenomenon, improves the compactness of the product structure, and results in a dense and juicy tissue protein structure [29]. Therefore, increasing the moisture content of the material is conducive to the promotion of protein-stretching denaturation, thereby improving the tissue protein quality of structural characteristics, including hardness, juiciness and other textural characteristics [41].
Moisture levels are also important for the production, storage and transportation of final products. In the most common extrusion process, for example, low moisture extrusion (with a moisture content of less than 40%) produces a relatively poor-tasting tissue protein, while high moisture extrusion (with a moisture content of more than 40%) retains moisture and produces a highly textured, elastic, tough, fresh and high-quality tissue protein with a texture similar to animal meat and an appearance and taste similar to cooked meat [42]. Furthermore, based on the difference in the moisture content of tissue protein, low-moisture tissue protein is less likely to spoil after drying and can be stored for a long time with a longer shelf life. However, the production cost of high-moisture tissue protein is high, and it has a short shelf life, with strict requirements for storage (during which it must be refrigerated) and transportation. Thus, further improvement is required.

4. Effects of Molding Process on the Structural Quality of Tissue Protein

In recent years, extrusion technology is still the main way to process tissue protein because it can form a fibrous structure similar to meat, and the research is relatively mature, in which high moisture extrusion has a better taste and gradually becomes the better choice. 3D printing technology is gaining the attention of scholars studying plant-based meat alternatives because of its personalization and nutritional customization and its ability to create products with complex structures or geometric shapes. Many scholars are gradually researching it in depth, expecting it to become an industrial production technology for plant-based meat alternatives [20][43][44]. Shear cell technology, spinning technology and refrigeration structure technology are also plant-based tissue-protein-processing technologies that distinguish themselves from the previous processing methods. In addition, the effects of different technologies on the structural quality of tissue protein are analyzed, and the characteristics of the finished products of different molding methods are presented (Figure 1).
Figure 1. Schematic diagram of the processing principle, process and finished products of different molding technologies [45][46][47]. (a) Schematic diagram of the processing principle, process and finished products of extrusion technology; (b) Schematic diagram of the processing principle, process and finished products of 3D printing technology; (c) Schematic diagram of the processing principle, process and finished products of shear cell processing technology; (d) Schematic diagram of the processing principle, process and finished products of electrostatic spinning technology; (e) Schematic diagram of the processing principle, process and finished products of refrigeration structure technology).

4.1. Extrusion Technology

Extrusion technology integrates the mixing, homogenizing, aging and forming of materials through thermomechanical treatment and shear flow to form a fibrous structure that is similar to meat. It is dependent on the moisture content of the material, and therefore, when the moisture content of the material is lower than 40%, it is a low moisture extrusion process, and if it is higher than 40%, then it is a high moisture extrusion process [42]Figure 1b is a graphical representation of the extrusion of final products with different moisture contents. The low moisture extrusion method does not require high protein content in raw materials [10]. Wet materials flow through the extruder at high temperature, the moisture in the material instantly turns to hot steam, the extrudate expands, and the structure becomes puffed sponge that must be rehydrated before use [48][49]. Low moisture extrusion is a more commonly used technology due to its wide applicability, high technical maturity and low equipment cost. High-moisture extrusion is a technique developed on the basis of low-moisture extrusion technology, which can better simulate the fiber structure of similar meat [50]. In the high-moisture extrusion process, the quality of the organized plant protein can be improved by increasing the protein concentration, and the high moisture content can ensure that the tissue protein will not break easily at high fiber strength, so the protein content of the raw material is usually more than 60%, and the tissue protein obtained via this process has higher moisture retention as well as high elasticity and toughness [51]. It is similar to meat products, with little loss of nutrients, and does not require rehydration before it can be eaten [52]. Due to its low energy consumption and excellent product performance, it is currently the most common method used in the production of juicy tissue protein in recombinant plant-based meat alternatives. While this technology and its market are still developing, the application potential of this approach is generally recognized by the industry as offering the most commercialization prospects [53].

4.2. 3D Printing Technology

3D printing technology, also called additive manufacturing technology for rapid prototyping, refers to the manufacturing of solid 3D structures created from virtual models through computer-aided design [54]. In this type of tissue protein molding technology, molten raw materials are squeezed out from a movable nozzle, solidified and then stacked, layer-by-layer, to obtain prints that resemble the structure of meat [55]. This technique requires the modification of matrix via different cross-linking methods and pre-treatment to obtain a plant-protein-based printing material that can be squeezed easily from the nozzle with sufficient mechanical strength [56][57]. Shahbazi et al. used 3D printing technology to produce plant-based meat alternatives based on soybean isolate protein and explored the effect of introducing biosurfactants to achieve partial fat replacement and improved structural properties of 3D printed products [58]. A team of researchers intend to improve the texture of alternative meat by directly inserting hydrocolloid-based fibers into the protein matrix using a coaxial nozzle-assisted 3D food printer [59]. Although the 3D printing of plant-based meat alternatives has been explored to some extent, it is not yet fully developed and requires further progress to achieve the immediate and rapid fabrication of structurally superior plant-based meat alternatives.

4.3. Other Technologies

Shear cell processing technology uses shear flow to mix raw plant protein materials and water. The processing time is controlled by adjusting the cylinder speed and temperature of the equipment. With anisotropic tissue proteins and a simple combination of shear force and heating, plant protein can be processed into homogenous, layered and multilayered fibrous structures [60]. Shear cell processing technology features a constant shear force and reduced mechanical energy dissipation, and is highly flexible in terms of required equipment. Moreover, it has great potential for growth in the field of tissue protein synthesis. The processed tissue protein has strong continuity in the shear direction, a long and slender fiber structure with a tight and dense interfiber, and is difficult to break [61].
Electrostatic spinning technology refers to the process in which nano-level oriented fibers are formed from plant protein polymer solutions in a nonwoven state under the breakdown of a high-voltage electrostatic field. However, the technological difficulty of this process is relatively high because proteins have a complex advanced structure, making them more difficult to destroy during spinning, and the pre-treatment requirements for raw materials are stringent [62]. These technical constraints limit the utilization of this method, as only some proteins are suitable for spinning technology, and, hence, it has relatively limited applications in tissue protein production.
Refrigeration structure technology produces porous fibrillar structures by, first, freezing protein emulsions and then removing the ice crystals to obtain tissue proteins that resemble the structure of animal muscle fibers and consist of many parallel and highly connected lamellar proteins [63]. The composite effect of the structural and physicochemical properties of different plant proteins results in differences in the structure of tissue proteins obtained from different plant-based composites. This technology has the capacity to form plant-based meat alternatives with unique textural contours and at a relatively low cost, making it suitable for small-scale industrial production [47].

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