Tissue engineering, a discipline established in the 1960s, depicts living organisms as assemblages of various tissues, each characterized by complex architectures that involves diverse cell types and extracellular matrices that serve distinct biological and biomechanical functions (Ikada, 2006; Vacanti, 2006). Although they focus mainly on biomedical applications for repairing living tissues, these principles can also offer a new lens through which to study and replicate the structure of livestock meat, a “product” comprising muscle tissue, connective tissue, adipose tissue, and bone (Ben-Arye and Levenberg, 2019) (Figure 1).

In its processed form, the elements of meat are intricately organized to form familiar elements or “products” such as steaks, which offer a unique culinary and organoleptic experience. Animal meat, with its inherent heterogeneous structure (such as the varied distribution of fat and muscle) and anisotropic characteristics (owing to muscle fibers and the orientation of connective tissue), presents a sophisticated challenge in food technology. The complex thermal transition during cooking, which is driven by the reactions of proteins and other biomolecules to heat, significantly influences its perceived quality and consumer experience (Tornberg, 2005; Yu et al., 2017). Previous attempts to replicate whole-muscle cuts with plant-based ingredients (Dekkers et al., 2018) often fell short of expectations, resulting in products with limited appeal to omnivores and minimal penetration beyond the vegetarian market (Szenderák et al., 2022).

The Plant-Based Tissue Engineering (PBTE) approach addresses these complexities by deconstructing meat into simpler elements, and then mimicking them using accessible food manufacturing technologies, before reassembling them using innovative fabrication methods inspired by Additive Manufacturing (Hertafeld et al., 2019) and robotics. This section describes each of the main tissue elements and its relevant characteristics, highlighting the aspects that are crucial to replicating the meat-eating experience. It outlines the sufficient structural elements and behaviors that are required for successful imitation of the organoleptic qualities of meat, but at the same time strives to limit the complexity of the reverse-engineered tissue components and their spatial resolution accuracy to avoid unnecessary development and production costs (GFI, 2022).

One of the guiding principles of our development process was that it would need to address a set of key characteristics to successfully imitate muscle components:

a. High protein content: This is essential if we aim to match the nutritional profile of meat.

It is important to mention, that this is a minimal set of characteristics providing the entry-point to steak-relevant texture. Further analysis of tensile behavior, shear behavior and sensory analysis is crucial for generating a product that will be accepted by consumers.

Current alternative meat production methods, such as protein extrusion (Areas, 1992; Dekkers et al., 2018), are inadequate to meet all the above requirements. TVP (Textured Vegetable Protein) (Emin and Schuchmann, 2017) and HME (High Moisture Extrusion) (Lin et al., 2000) techniques fall short in mimicking all the above requirements to replicate the specific texture and structural integrity of meat. TVP is too porous and HME is too dense and impermeable, and neither can be formed into a steak form factor (e.g., at least 150 × 80 × 15 mm), while providing relevant orientation of fibers. Methods like shear cell (Krintiras et al., 2016) address the dimensional requirements yet fall short in providing beef-relevant texture. Other techniques, such as spinning (Mattis and Marangoni, 2020), fail to provide sufficient throughput at an acceptable cost. Bioengineering approaches, such as mycelium (Kyoungju et al., 2011) or animal tissue culture (Post, 2012) still lack the required texture demands - and require supplementing them with plant-based texture elements (Ben-Arye et al., 2020), but even then, they fail to deliver the relevant meat performance operating in industrial environments (Post et al., 2020).

To meet the requirements of the muscle component, our research proposes disintegrating the tough and chewy TVP material into separate fibers (0.2–2 mm in diameter, about 2–20 mm in length), blending them with a proteinous dough made from soy or pea isolates (Ben-Shitrit et al., 2021). A typical TVP product suiting the toughness criteria typically comprises about 50% wheat protein and soy protein and has relatively low (<250% w/w) water holding ability, such as DuPont Danisco SUPRO^® MAX 5050. This dough, when extruded using a progressive cavity pump (PCP, PCM Ecomoineau™ C) through a 3 mm nozzle, delivers strands that imitate the hierarchical structure of fibrous livestock meat, namely, stacked fascicles (Ben-Shitrit et al., 2021). To form a macroscopic muscle section, the deposition is made according to typical 3D-printing protocols, in a self-supporting manner, as the dough viscosity is sufficiently high (>300 P) to prevent gravitational flow or sagging at the fabrication temperature that is set to about 4°C to meet food safety requirements. Unidirectional deposition of such strands results in structure having prominent anisotropic behavior and its resistance to shear being comparable to that of animal meat. This approach allows the projection of the meat-like texture of TVP, but in a flexible manner, and in a scalable manner. At the same time, this muscle-like structure alone is insufficient in meeting livestock meat’s tensile behavior values and fully mimic its inter-fascicle failure mechanisms. This deficiency required the development and introduction of a component that would act as connective tissue and reinforce the composite structure of the meat alternative and upgrade its toughness and behavior during cooking and eating (such as the disintegration of fibers) in a way that matches the behavior of animal meat.

Developing a connective tissue (CT) component posed a few significant challenges:

a. Obtaining Food-Grade Ingredients and Processes: Using ingredients and formulations that are safe and compliant for consumption, while meeting the structural requirements listed below. Most plant-based edible materials lack the strength that is characteristic of animal tissues.

We found that carrageenan-based materials with melting temperatures around 90°C fulfil these requirements and could be cast into films, which are then introduced in-between the fascicles during layer-wise fabrication (Figures 2A,B), while setting one step interlace between the strands (Dikovsky and Hausner, 2022). Post-hydration and thermal processing result in fusion of the carrageenan films and formation of continuous comb-shaped network (Figure 2C), with tensile strengths mimicking that of animal meat. Table 1 compares the tensile strength of meat measured along and across the direction of the fibers. It shows that the addition of the CT component substantially improves the performance of PB meat, matching its strength to that of a tenderloin (Psoas Major) at 90% at longitudinal direction and 85% at transverse direction. Moreover, the plant-based muscle with CT demonstrates a different failure mode upon stretching (Figure 3A), that is comparable to livestock meat (Figure 3B) and substantially different from plant-based muscle without CT (Figure 3C). Internal sensory panel tests (n = 30) indicated 82% preference for alternative meat samples that contained CT compared to those without CT, with specific feedback from participants noting improvements in texture and appearance. The characteristic fibrous texture of such meat is shown after dissection in Figure 2G.

The muscle component and connective tissue component are integrated into a system that employs additive manufacturing methods accompanied by robotic film deposition, altogether digitally controlled, to deliver about a variety of alternative meat muscle tissue structures. The following parameters can easily be adapted to control the toughness, fibrousity, chewiness and cooking behavior of the alternative meat:

• Type of TVP

The final last component in the list of components is fat, and fat distribution and marbling.

The fat component, crucial to achieve the desired texture and mouthfeel, involves encapsulating a lipid formulation (melting point >35°C, using primarily cacao fat) in a hydrocolloid matrix. This matrix, composed of methylcellulose, stabilizes the fat tissue during cooking and prevents it from melting out. The high lipid content (≥75% w/w) ensures a substantial liquid oily phase at serving temperature (50°C), contributing to a fatty mouthfeel. The different types of fat, such as intermuscular fat and intramuscular fat, can be recreated, for example, via the variation of overall lipid content and the concentration methylcellulose in the matrix. This fat component is compatible with the additive manufacturing process, allowing precise deposition to recreate specific marbling patterns. Here too, the viscosity of the fat component paste is maintained high enough to allow self-supporting deposition using typical PCP pumps. Due to the nature of marbling in beef steaks, the fat deposition resolution may require thinner nozzles, down to 1 mm. The interconnection between the muscle component and the fat component is facilitated by the presence of the CT component that holds together the unidirectional elements that form the muscle and fat tissue regions.

Breaking meat down into components and addressing each component separately allows more accurate mimicking using plant-based ingredients. However, the assembly of these components into a complete meat product requires new approaches to food manufacturing. Inspired by Additive Manufacturing (AM) and robotics, we developed a process that uses high-pressure extrusion devices and a static multi-nozzle array, delivering high-viscosity component pastes onto a digitally controlled XYZ platform (Ben-Shitrit et al., 2020). Other elements of the plant-based meat, such as the CT laminates, are dispensed via dedicated robotic modules. The use of this advanced manufacturing method enables the creation of any spatial configuration of muscle and fat, supporting the production of relevant whole-muscle cut analogs.

The Additive Manufacturing (AM) process facilitates:

• Spatial configuration control of muscle and fat and realistic marbling.

While successfully exploiting the benefits of AM, it was important to address its major weakness points, namely, cost and throughput, that frequently limit its implementation in industrial applications and could impact the market potential of the current solution. This was achieved through a design-for-function approach, where the fabrication system was optimized solely for beef whole muscle cuts application, while maximizing throughput. In practice this resulted in a system operating at relatively coarse spatial resolution, depositing about 3 mm strands, with no need for support material or curing, with all strands being laid in the same orientation forming long slabs, approximately 1-m in length. Altogether, this resulted in minimal tray idle time, maximized travel velocity and low waste. Once combined with a 12-nozzle plate simultaneously depositing material, the system generated a throughput of more than 10 kg/h, an equivalent of about cow a day of alt-meat products (Mandelik et al., 2021).

Figure 3E shows a set of animal-free whole muscle cuts made using the PBTE concept and matching some of the common premium cuts, such as tenderloin, sirloin, ribeye and wagyu. All of these were cut from slabs made with the same set of muscle, fat and connective tissue components and digitally manipulated to form the desired form factor and marbling patterns.

The PBTE approach represents a significant contribution to the field of Soft Matter, particularly in food science. It showcases how the manipulation of plant-based materials at the microstructural level can lead to the development of complex, multi-component food systems. By mimicking the hierarchical structure of muscle, adipose, and connective tissues in meat, we can bridge a crucial gap in the alternative protein industry, aligning with the Soft Matter principles of understanding and engineering complex structures and materials.

• Muscle Tissue Replication: Through disintegration of TVP into fibers blended with proteinous dough, we successfully mimicked the anisotropic and fibrous structure of muscle tissue. The introduction of a connective tissue component further enhanced the mechanical integrity and sensory attributes of the muscle analog, closely resembling those of animal meat.

• Environmental Impact: Given the resource-intensive nature of traditional meat production, the PBTE method offers a more sustainable alternative to meat, potentially reducing greenhouse gas emissions and the use of resources like water, land, and energy.

In conclusion, the PBTE approach marks a pivotal step in the evolution of meat alternatives. By intricately replicating the structure and texture of animal meat using plant-based ingredients, we not only address environmental and ethical concerns but also cater to the growing demand for sustainable and diverse protein sources that meet consumer expectations for taste, texture and other parameters of meat. The principles and methodologies developed in this study hold significant potential for future innovations in the Soft Matter field, particularly in helping to develop sustainable food systems.