Meat and meat alternatives: where is the gap in scientific knowledge and technology?

Abstract

The unprecedented surge of developing meat analogues and other alternative protein products in the past few years has occurred amidst numerous technological challenges. The creation of fibril structure and flavour from non-muscle proteinaceous sources to mimic fresh and processed meat requires a clear understanding of the fundamental differences between muscle and alternative proteins. Currently, plant-based meat alternatives are dominating the alternative market; mycoprotein-based products have also gained a market share, while algae and insects are other emerging sources of alternative proteins. Both traditional and novel processing technologies and innovative ingredient formulations are being developed to transform nonmuscle proteins into fibrous or particle structures that microscopically resemble muscle. However, the production of meat analogues generally entails ultra-processing and ultra-formulation, which could compromise nutritional value and safety. Therefore, to sustain the alternative protein market, scientists and entrepreneurs must methodically examine and understand inherent differences that separate alternative proteins from muscle proteins. An intuitive knowledge base is essential to designing new approaches to overcome technical challenges. In doing so, food scientists and entrepreneurs must be keenly cognisant that animal meat products are unique; the quality and sensorial attributes of meat can only be simulated but not replicated.

Keywords:

• Meat
• alternative proteins
• meat analogues
• restructuring

Introduction

Meat analogues and other imitation or substitution products, categorically referred to as ‘meat alternatives’ or ‘alt-meat’, are a rapidly developing market that has attracted both traditional vegetarians and an increasing number of omnivores. The latter group, including flexitarians who strive to maintain a balanced diet, represents the principal population of consumers worldwide who enjoy the meat-eating experience while being cognisant of nutrition and health. Meat alternatives currently constitute a USD 9.9 billion market (or 5% of the ‘meat’ sector), and the burgeoning industry is projected to expand at a compound annual growth rate of 42.1% to reach USD 234.7 billion by year 2030 (Research and Markets 2022). The main driving forces for this emerging market are multiple, including food security and sustainability, environmental protection, animal welfare, and health. Although plant-based processed food is widely consumed worldwide, in Western countries and many other parts of the world, there is a general lack of variety of structured products made from nonmuscle proteins. Therefore, it seems to make logical sense to develop novel foods that structurally and organoleptically resemble traditional foods that consumers are familiar with. Meat mimetics made from alternative proteins using restructuring and ingredient technologies are one of those innovations (Sha and Xiong 2020).

Meat alternatives have been prepared from a range of protein-rich edible materials, including legume seeds (pulses), fermentation-based fungi, as well as microalgae and insects (Van der Weele et al. 2019). More than 90% of meat alternatives available in the global market are manufactured from plant protein sources, for example, soy burgers, pea sausages, and lentil nuggets. Fibrous cereal proteins (glutens) and polysaccharides are commonly included to help with restructuring or improve product texture. In addition, considered as innovation revolutions, in vitro cultivation of muscle stem cells (Choudhury et al. 2020) and 3D printing (Ramachandraiah 2021) have been introduced to produce animal-free meat and meat-like steaks and chops, respectively.

Global consumer acceptance and attitude towards meat analogues vary widely, depending on the region, culture, lifestyle, and age of the consumer (Onwezen et al. 2021). For example, in traditional meat-production and meat-consumption countries, including the United States, most South America countries, China, Italy, and Finland (to name a few), consumers continue to enjoy meat while adapting to the niche market of plant-based alternatives. Yet, the indifference and unwillingness to accept nonmeat products as total or partial substitutes for regular animal meat remains prevalent and will likely continue in the near future.

Aside from the reluctant attitude towards drastically reducing meat consumption in a large part of the world, there are major technical hurdles and impediments in the development of meat analogues and mimetics. There have been tremendous efforts to create fibrous protein structure from non-meat sources that mimic muscle utilising traditional thermoplastic extrusion and spinning, as well as conical shear cell technology, freezing, and protein-carbohydrate aggregation (Dekkers et al. 2018; Wang et al. 2023). However, the structure of muscle is extremely complex and hierarchical in nature, and endogenous constituents responsible for the unique texture, mouthfeel, and flavour of meat (protein, lipid, nucleotides, minerals, vitamins, pigments, and other organic flavour compounds) are compartmented within the well-structured micro- and nano-scale fibril or myofilament lattices. It is therefore formidable to attempt to recreate such fine structural features that impart the one-of-a-kind taste experience of real meat. Due to various technical hurdles, there has not been a single technological intervention so far that can be considered a total success in constructing a meat-like product despite much of the effort. The difficulty to recreate meat texture and flavour continues to impede the alternative meat market development which presumably contributes to its stagnant growth and even setback seen in 2022 (Buss 2023).

This brief review will describe the structural and molecular differences between muscle and alternative proteins and highlight recent engineering and physicochemical approaches to creating muscle fibril-like structures and functional products (gels, emulsions, aggregates, etc.) intended for meat analogues. Instead of detailing individual studies, the review will focus on categorical similarities and differences between animal meat and alternative products providing a broad landscape of the alternatives market. Legume (pulse), cereal, fungal, microalgae, and insect proteins are included in the discussion for their established or potential use in meat analogue production. Emerging bottom-up strategies, including cell cultivation and 3-D printing, are not specifically discussed due to their limited market impact at the present time and preliminaries in product development. Current understanding of protein molecular interactions, and the lack thereof, in building the muscle-like texture will be highlighted. By addressing the knowledge gap and technological voids that separate muscle and alternative products, the author hopes the review could offer useful guidance in the on-going and continuing effort to grow the alternative protein market that ensures a sustainable supply of nutritious and palatable food.

Muscle structure and protein constituents

Because muscle is the ultimate target that is to be simulated in alternative products, it is important to describe the general structural and processing characteristics of muscle providing the necessary context. Muscle is a highly structured apparatus that is organised in four levels of hierarchical units: bundles of fibres, individual fibres, myofibrils, and myofilaments (Figure 1). The entire muscle is surrounded by a sheath of connective tissue (epimysium) that provides an overall structural protection. The fibre bundles, made up of individual fibres, are encased by another layer of connective tissue (perimysium). An individual muscle fibre (cell) is stabilised by endomysium connective tissue and consists of segments (sarcomeres) each comprised of several hundred fine myofilaments that run along the length of the myofibril. The thick (myosin) and thin (actin) filaments are 16–17 and 6–7 nm in diameter, respectively, and they are interconnected to generate three-dimensional micro- and nano-structural lattices (Matsuda and Podolsky 1986). Moisture, micronutrients, pigments, and other cellular components are confined within the structural lattices. Organoleptically, it is the penetration of the teeth across the muscle fibres and myofibrils that produce the sensorial experience and mouthfeel of cooked meat upon mastication. Although the bulk of water is well bound by capillarity and entrapped within the muscle cell, it is readily released during mastication providing sensorial juiciness. However, the lack of such delicate, highly structured myofibril apparatus and confining membrane in restructured nonmeat alternative products makes it virtually impossible to reproduce the moisture releasing experience.

Figure 1. Skeletal muscle structure showing a characteristic hierarchical orientation and major protein constituents. From essentials of anatomy and physiology. https://www.brainkart.com/subject/Essentials-of-Anatomy-and-Physiology_259/.

The structure of skeletal muscle is highly conserved although there are minor variations between mammalians (beef, pork, and lamb), poultry, and fish species. The principal proteins comprising the muscle fibre are myosin (500 kDa) and actin (45 kDa). In whole-muscle meat, the actomyosin complex and its integrity are crucial to the texture (e.g. chewiness and mouthfeel) perceived by the consumer. In processed meat products, such as hamburgers, sausages, frankfurters, and structured ham, the functionality of actomyosin and myosin extracted by salt and phosphate, i.e. gelation, emulsification, and water-holding, are responsible for texture-related sensory attributes (e.g. tenderness and juiciness). Salt-dependent myofibrillar protein solubilisation and subsequent thermal gelation of myosin and actomyosin are essential for the adhesion of meat particles as well as the stabilisation of emulsified fat and entrapment of water (Sun and Holley 2011; Xiong 2017).

Alternative proteins

Legume and cereal proteins

While muscle structure and proteins are largely conserved, plant proteins are heterogenous and of vastly different structures and properties. For legumes (Fabaceae), the pulses (seeds of beans, peas, and lentils) are rich sources of storage proteins. The major storage proteins found in soybeans, peas, mung beans, lupins, lentils, and many other legume crops are 11S (legumin-type) and 7S (vicilin and convicilin-type) proteins (Derbyshire et al. 1976). Because of the globular structure, pulse proteins must be processed into a fibrous form or shape to mimic muscle fibrils unless the product is a comminuted (i.e. chopped) meat analogue (Table 1). For cereals (wheat, corn, rice, oat, etc.), the solubility-based protein types are extremely heterogenous. For example, oat and rice are abundant in water- or salt-soluble globulins, while wheat and most other cereal grains are rich sources of ethanol-soluble prolamins and alkaline-soluble glutelins (Shewry and Halford 2002). These gluten polypeptides are fibrous in shape, rich in glutamine and proline, and suitable for meat alternatives by supporting the product structure.

Table 1. A generalised description and comparison of meat and alternative products.

Product category	Processing	Additives	Product characteristic	Nutrition	Further reading
Whole-muscle meat	Sectioned muscle; minimal processing; tumbling and massaging (boneless hams, bacon, corned beef;…)	Additive-free for fresh meat; otherwise salt (2–3%); phosphate (0.3–0.5%); nitrite (120–200 ppm) and other curing agents; …	Typical meat colour, flavour, tenderness, chewiness, and mouthfeel; juiciness; …	Complete amino acids; rich source of Fe, Cu, Zn, vitamin B1; varying amounts of saturated fat and cholesterol	Toldrá 2022.
Ground meat	Grinding, mixing, and shaping into different products (burgers, fresh, processed or fermented sausages, …)	Additive-free for fresh burgers; salt (2–3%) for sausage and patties; plant protein extenders; seasonings; …	Typical meat colour and flavour; juiciness; …	Complete amino acids; rich source of Fe, Cu, Zn, vitamin B1; varying amounts of saturated fat and cholesterol	Ahmed et al. 1990; Suchenko et al. 2017
Comminuted meat	Finely chopping and emulsifying into batters	Salt (2–3%); phosphates (0.3–0.5%); plant protein extenders; nitrite (120–200 ppm) and other curing agents, seasonings; …	Typical meat or nitrite-cured flavour, colour, tenderness, and mouthfeel; juiciness; fine texture; …	Complete amino acids; rich source of Fe, Cu, Zn, vitamin B1; varying amounts of saturated fat and cholesterol	Claus et al. 1990; Bolger et al. 2017; Xiong 2017
Fibrillised pulse-based meat analogue	High-moisture extrusion to form anisotropic protein fibres; crosslinking to texturise	Adjunctive wheat gluten and polysaccharides; transglutaminase; salt (varying amounts); colourants; seasonings; …	Well-textured; good mouthfeel; varying juiciness; generally weak meaty flavour; low meat-like chewiness	Nutrition can be improved with supplemental nutrients (vitamins and minerals); low in saturated fat but also low in methionine	Van der Weele et al. 2019; Wang et al. 2023
Pulse-based coarse-particle meat alternative	Low or high-moisture extrusion for fibres or texturisation; fabrication for desirable particle size	Polysaccharides, vegetable oil; salt; colourants; seasonings; vitamins and minerals; …	Good flavour and mouthfeel; weak particle-binding; bound water not readily released upon mastication	Nutrition can be improved with supplemental nutrients (vitamins and minerals); low in saturated fat but also low in methionine	Davis et al. 2021; Zhou et al. 2022
Mycoprotein-based fine-particle meat alternative	Fungal fermentation to obtain mycelial protein; blending and compression into structured analogues	Vegetable oil; salt; colourants; seasonings; vitamins and minerals; …	Smooth texture and mouthfeel; spongy	Nutrition can be improved with supplemental nutrients (vitamins and minerals); potential to decrease serum low density lipoprotein	Hashempour-Baltork et al. 2020; Cordelle et al. 2022
Microalgae-based meat alternative	Extrusion or simple mixing and compression into structured analogues	Pulse protein (co-extrudate for fibrillary texture); salt; colourants; seasoning (to mask odour and colour); vitamins; …	Shear force and hardness vary with formulation and moisture content	Nutrition can be improved with supplemental vitamins	Caporgno et al. 2020; Fu et al. 2021
Insect protein-based meat alternative	Requiring co-extrudate proteins for structure formation	Pulse and cereal protein (co-extrudates); colourants; seasonings (to mask odour); salt; …	Texture and mouthfeel vary depending on products (jerkies, sausage, etc.)	Protein quality (essential amino acids) varies extensively; good digestibility; nutrition can be improved with supplemental vitamins	Smetana et al. 2018; Scholliers et al. 2020; Kim et al. 2022; Kurek et al. 2022

Differing from muscle proteins, plant proteins are rather complex and generally consist of multiple subunits. Isolated plant seed storage proteins are always found as complexes, aggregates, or agglomerates, and disulphide bonds are prevalent in the protein structure. Some plant proteins are also naturally associated with carbohydrates, lipids, polyphenols, and other chemical compounds. Therefore, the extraction and preparation of proteins from their host plant seeds require vigorous cell wall disruption for separation, enrichment, and purification. Both wet extraction and dry fractionation are employed to isolate proteins. The former method generally involves the solubilisation of proteins with an alkaline aqueous medium followed by isoelectric precipitation to recover the protein (Hadnađev et al. 2017). Dry fractionation involves the rupture of seeds through fine milling and subsequent pneumatic classification to obtain crude proteins (Fernando 2021). To facilitate protein extraction, adjunctive technologies can be applied, for example, microwave, ultrasound, and ohmic heating.

Microalgae proteins

Microalgae are a vast group of marine ‘plants’ encompassing some 200,000 species. Natural and cultivated microalgae generally have a very high protein content with most ranging from 40% to 60% (Becker 2007). Spirulina and chlorella, two dominant species in the alternative protein market, have a reported protein content up to 77% (Koli et al. 2022). The extremely high protein content combined with the huge biomass makes microalgae an attractive source of protein for meat alternatives. Moreover, the essential amino acids index of some microalgae is high and comparable to animal source proteins (Kent et al. 2015). Algae proteins are extracted after cell wall disruption and separated by centrifugation into soluble and insoluble fractions; proteins in the soluble fraction are recovered by isoelectric pH precipitation (Böcker et al. 2021). To facilitate extraction, alkaline aqueous solutions may be used, similar to pulse protein extraction. There is a broad size distribution for microalgae proteins. For example, in spirulina extract, which is comprised of more than 50 detectable proteins by SDS–PAGE, the molecular weights range from 2 to 250 kDa (Zhou et al. 2021). Yet, currently, there is scant information on the structural and physicochemical properties of microalgae proteins or fractions. Nevertheless, microalgae proteins are shown to be surface-active, capable of stabilising oil-in-water emulsions for potential application in formulated alternative products (Bertsch et al. 2021).

Mycoproteins

Microbial proteins are single-cell proteins cultivated from bacteria, yeasts, and moulds through fermentation. Mycoproteins, also known as fungal proteins, are derived from fungi, which have been successfully used as the building block of meat alternatives. Branded as Quorn, the mycelia protein produced by Fusarium venenatum A3/5 fungus through fermentation is by far the most successful commercial mycoprotein source in the alternative protein market. The filamentous fermented protein is bound with egg albumin or plant extract then steamed and chilled. The structured mycelia mimic muscle fibres and impart the meat-like mouthfeel and texture. Hence, thermoextrusion and other fibrillisation processes are not required.

Both filamentous and globular proteins and their aggregates are produced from fungal fermentation. The polypeptide composition of mycoproteins is extremely complex and heterogenous; therefore, they are difficult to characterise. Based on SDS-PAGE analysis, hundreds of individual proteins and peptides with molecular mass ranging from several kDa to greater than 200 kDa are present in the extracts of fermented biomass (Colosimo et al. 2020). Although only a handful of individual mycoproteins have been studied, mixed myoproteins have been shown in model systems to have excellent gelling, emulsifying, and foaming activity (Lonchamp et al. 2022). These functional properties, together with the filamentous structure and ability to coagulate, contribute to the structure-building potential in texturised meat alternative products. Myoprotein-based products available in the market include analogues of nuggets, burgers, fish fillets, and deli meats.

Insect proteins

An insect is a small arthropod animal that, depending on the life cycle stage, does or does not have six legs and one or two pairs of wings. Insects represent an extremely diverse group of alternative protein sources. More than 2000 insect species are considered edible; this includes embryo, larva, and adult stages. The production, processing, innovative food and feed application, nutrition, and safety have been extensively studied and documented (Rumpold and Schlüter 2015). Insects are considered valuable sources of proteins for meat alternative, due to their relatively high protein content (up to 40–70%) and easy production. Because of their distinct flavour (off-flavour) and consumers’ neophobia, insects as food are usually processed into powder to destroy their morphology and incorporated into formulated food as additives (cookies, bakery products, etc.) rather than sold as a whole and stand-alone product. Alkaline aqueous extraction after disruptive pre-treatment is applied to prepare concentrated proteins with desirable functionality.

The functionality of insect protein varies depending on the type of insects. For example, water and fat absorption capacities of Hermetia illucens (black soldier flies, a high protein insect with a 42% protein content) were reported to be 1.3 and 3 g/g, respectively (Mintah et al. 2020). Proteins extracted from Tenebrio molitor, Schistocerca gregaria, and Gryllodes sigillatus exhibited comparable emulsifying properties to legume proteins (Zielińska et al. 2018). The gelling capacity, another important functional property for alternative proteins intended for meat analogues, has barely been investigated. Yi et al. (2013) evaluated the gelling potential of the protein extracts from Tenebrio molitor, Zophobas morio, Alphitobius diaperinus, Acheta domesticus, and Blaptica dubia reporting that gelation at pH 7.0 was possible but a very high protein concentration (e.g. 30%) was required. Although insect proteins may be of limited functionality when used alone, they can be combined with meat proteins to produce an adhesive co-gel suitable for supplemental meat products (Scholliers et al. 2020). Similarly, insect proteins can be combined with plant proteins to produce meat analogues. Kim et al. (2022) reported that a homogenate of mealworms (22.7% protein)/textured vegetable protein (TVP) could be processed into dry jerky analogues.

Reconstruction of muscle-like structure from alternative proteins

In terms of structure, meat alternatives can be divided into two general groups: (1) whole or intact muscle-like analogues comprised of fibrils and (2) particle-based restructured products. The latter is further divided into two subgroups, i.e. coarse-particle and fine-particle (emulsion) products (Figure 2). Most commercial meat alternatives sold in the current market are coarse particle-based that texturally resemble the structure and mouthfeel of ground meat, for example, soy burgers, pea sausages, and insect nuggets. Fine particle-based alternatives include gelled emulsion products that mimic the smooth texture and sliceability of regular frankfurters and many deli-type luncheon meats. In these products, protein fibrillisation is generally not necessary because gelation and the formation of a fine protein matrix are the determinant factors for product texture.

Figure 2. Skeletal production of meat alternatives from nonmuscle proteins. Note the three types of meat analogues: whole muscle-like (1), coarse-particle type (2), and fine-particle emulsion type (3).

Whole muscle-like products

Thermo-mechanical extrusion is by far the most developed and widely adopted technology to transform nonmuscle proteins into structured aggregates or fibres suitable for subsequent fabrication into the whole muscle-like texture (Sun et al. 2022). Extrusion at high temperatures (120–180 °C) has long been used to process soy-based TVP as fillers in sausage or as ingredients in other food products. The traditional low-moisture extrusion (20–35% moisture) process has been applied in the processing of legume proteins into a structured base material for alternative products, including plant-based hamburgers and sausages. On the other hand, high-moisture extrusion (40–70% moisture) processing is a more suitable option due to its ability to produce muscle-like fibres under appropriate heating, shearing, and cooling conditions (Table 1). The operation involves multiple steps, i.e. mixing, hydration, shearing, homogenisation, compression, deaeration, heating, molecular crosslinking and alignment, and shaping (Zhang et al. 2022). Protein ingredients in the extrusion process are subjected to micro-coagulation and fibrillisation with the procedure being carefully controlled to produce anisotropic fibres. Wheat gluten and carbohydrate polymers are often added as copolymers to facilitate fibre formation and alignment. High-moisture extraction technology has been applied in the processing of plant-based alternatives (Dekkers et al. 2018) as well as insect-based (Smetana et al. 2018) or algae-based (Caporgno et al. 2020) meat analogues. In addition, several other technologies have been developed to fabricate fibres of nonmuscle proteins, including wet spinning, electrospinning, and conical shear. These techniques are still under development, and the structural properties of resulting fibres have been reviewed (Dekkers et al. 2018; Wang et al. 2023).

Microstructurally, extrusion-generated fibres from plant, insect, and microalgae proteins lack the high-order structural alignments and well-defined nanoscale fibrillar texture exhibited by muscle fibres (Figure 3). Inherently, the size of a myofibril (1–2 µm diameter), which comprises muscle fibres and cells, is rather constant, and the interstitial space between thick filaments (myosin; 16–7 nm diameter) and thin filaments (actin; 6–7 nm diameter) is on a nanoscale. It has been shown that extrusion is limited to forming fibrous structures on microscale with a fibre diameter of about 50 μm (Dekkers et al. 2018). It is noteworthy that in order to recreate a muscle fibre-resembling structure, the high temperature (up to 180 °C) extrusion condition followed by cooling unavoidably generates tightly associated aggregates stabilised by random hydrophobic interaction and covalent bonds. This differs from animal myofibers and myofibrils in which ionic interactions and hydrogen bonds are the predominant stabilising forces and covalent bonds are absent (Xiong 2017). The rather moderate cooking temperature (60–80 °C) does not dispose muscle to significant fibre aggregation and shrinkage (Figure 3). Hence, it is virtually impossible to recreate the type of complex hierarchical organisation of muscle fibres let al.one the attainment of regular myofibrillar interstitial spaces. Yet, the interconnected sarcomere segments in muscle, consisting of longitudinal myosin thick filaments, actin thin filaments, and titin filaments, are the location where moisture is entrapped to impart the juicy organoleptic properties in cooked meat.

Figure 3. Micrographs of high moisture-extruded meat analogues made from lupin protein (A; Palanisamy, Franke, et al. 2019) and insect protein (B; Smetana et al. 2018), in comparison with raw (C-a) and stewed (C-b; 100 C 30 min) beef (Kaur et al. 2014) or raw (D-a) and immersion-cooked (D-b; 70 C 30 min) beef (Zhu et al. 2018). Modified from the cited studies.

To alleviate the harsh extrusion condition for micro and nanostructure mimicking muscle fibres, one possible strategy is to modify the source proteins through structure-modifying pre-treatments, such as high pressure, ball mill, cold plasma, ultrasound, and fermentation (Nasrabadi et al. 2021). The structure-based approach would allow the input of moderate and less destructive thermo-pressure conditions for fibrillisation, thus, enabling better control for anisotropic alignments, cross-linking, and aggregation of protein molecules. In addition, a holistic approach to optimising the combined interaction of the moisture content, pH, ionic strength, and cross-linking agents should be vigorously explored. Further research that combines structural chemistry and engineering design would be required to fill the knowledge gap.

Particle-based products

This group of meat alternatives is essentially a type of restructured products in which molecular proteins behave chemically and physically to form a desirable texture and bind water within a fabricated food matrix. The group can be subdivided into: 1) coarse-particle products, and 2) fine-particle products (Table 1). The vast majority of meat analogues in the global food market belong to this group and are plant-based, e.g. soy burgers, pea nuggets, and lentil sausage patties. To obtain a cohesive texture, methylcellulose and other polysaccharides are commonly included to help bind meat and fat particles and increase thickness through gelation (Bakhsh et al. 2021). The mixture of pea protein and apple pectin was reported to also have a strong binding capacity and may be used as an adhesive agent (glue) for sausage and hamburger-type alternatives (Moll et al. 2022).

Several studies have compared commercially available animal meat products with plant-based alternatives with regard to physicochemical characteristics and sensory properties. According to a recent report (Davis et al. 2021), plant-based retail ground beef alternatives, in comparison with real ground beef at 10, 20, and 27% fat levels, showed significantly lower instrumental shear force and red colour intensity and were consistently rated lower in appearance, juiciness, beefy flavour, and tenderness but higher in odour by consumers. In corroboration, Zhou et al. (2022), who compared five types of commercial beef burgers with five types of plant-based burgers, found that plant-based meat analogues had inferior rheological properties (soft, less cohesive, and low in chewiness) than meat burgers; the strong water binding (low cooking loss) by the added polymer ingredients would explain the dryness (less juicy due to poor moisture release) commonly observed in alternative products.

Vegetable oils are added to particle-type meat analogues to offset lost juiciness associated with the fat removal. Because of the solid nature, solid animal fat can be readily chopped into smaller particles and stabilised through emulsification by salt-extracted myofibrillar protein (MP). For meat analogues, however, unless fully hydrogenated oils or ‘saturated’ oils (coconut oil, palm oil, etc.) are used, vegetable oils as a fat replacement cannot be effectively immobilised within the viscous batter and require pre-treatment to ensure stability. Immobilisation of fluid oils for proper incorporation into the protein batters can be achieved through a number of techniques, including emulsification and microencapsulation. For example, the formation of the so-called Oleogel, which entraps oil and has the potential for emulsion-type meat alternatives, is accomplished by means of ethylcellulose when it is dispersed in oil at temperatures above its glass transition point and subsequently cooling to form a polymer gel (Davidovich-Pinhas et al. 2014). In addition, as described by Dreher et al. (2020), vegetable oil particles may be prepared by emulsification with plant proteins and subsequently cross-linked by transglutaminase (Figure 4). Insect protein powders prepared from house cricket were added as a protein supplement in meat emulsions (Kim et al. 2017). However, the fortified sausage had a reduced water-holding capacity and increased hardness. In other insect-based meat analogues, co-extruded Alphitobius diaperinus (lesser mealworm) protein and soy protein showed a good fibrous texture with moisture retention improved by the presence of soy protein (Smetana et al. 2018). Proteins extracted from many microalgae species also show good interfacial and emulsifying properties, with some being comparable to animal or plant-based proteins (Bertsch et al. 2021). Due to their low isoelectric point, microalgae protein-stabilised emulsions exhibited minor pH dependency and were resistant to changing ionic strength.

Figure 4. Schematic drawing of structural and property transitions of dispersed, concentrated lipid systems upon increases in solid fat content or enzymatic crosslinking. From Dreher et al. (2020).

For comminuted, finely textured products, protein gelation plays an essential role in product structure formation, stabilisation, and sensorial rheology. In muscle-based foods, solubilised myofibrillar proteins in the presence of 2–3% salt readily form an isotropic gel when cooked to 65–75 °C then chilled. The glue-like gel is adhesive, binds fine meat particles together, and immobilises fat droplets and water (Xiong 2017). Few alternative proteins exhibit such strong gelling potential. For example, soy and pea globulins obtained by alkaline extraction are able to gel only at high concentrations (generally 12–14%), and the gels are generally weak and lack the elasticity when compared with myofibrillar proteins, which can gel at 1% concentration (Lee et al. 1997; Nicolai and Chassenieux 2019). To improve their gelation, alternative proteins can be subjected to a number of structure-modifying pre-treatments that promote aggregation and cross-linking upon subsequent heating. Some of the most successful means include pH shift (Jiang et al. 2018; Ma et al. 2022), atmospheric cold plasma (Zhang et al. 2021; Basak and Annapure 2022), and high-intensity ultrasound (Sha et al. 2021).

While pulse and cereal proteins have been extensively studied, there is limited research on the functional properties and technological details for insect, fungal, and algal proteins. For practical purposes, in many food applications insects are utilised as dry powders other than protein concentrates or isolates. Therefore, it is rather difficult to attribute functionality of insect-based alternatives to any of the protein components. Yi et al. (2013) reported that at a very high concentration (30%), the soluble fraction of the proteins extracted from five insect species (mealworm larvae, superworm larvae, buffalo worm larvae, adult house crickets, and adult dubia cockroaches) could form a gel at pH 7.0. Purschke et al. (2018) demonstrated that the soluble fraction of locust protein concentrate had excellent emulsifying activity, comparable to egg albumen. For microalgae proteins, some exhibit excellent surface activity and are able to stabilise oil-in-water emulsions suggesting the potential for fine-textured meat analogues (Bertsch et al. 2021).

Flavour and appearance

Flavour (taste and aroma) is another significant challenge in the development of meat alternatives; at present, there is a lack of knowledge and technical expertise to successfully regenerate meat flavours. The flavour of meat is extremely complex and rather unique. Despite species-inherent variations, meat aroma and taste are distinct separating muscle foods (red meat, poultry, and fish) from any other commodity foods. Contributing to the overall flavour of meat are both water-soluble substances, such as nucleotides, haem proteins, peptides and amino compounds, as well as fat-soluble compounds that are deposited in the animal adipose tissue (Dwivedi and Brockmann 1975). For plant and other alternative protein sources, their metabolic systems drastically differ from animals, and each is inherently programmed. For example, legume proteins have the typical beany flavour, algae proteins are often perceived as fishy, and insect proteins have variable species-specific odours. The flavour challenges may be partially overcome through formulation innovation by adding impact volatile and non-volatile ingredients that either mask strong endogenous off-flavours or mimic meat flavour. For extrusion-processed proteins, the over-cooked flavour presents an additional impediment to meat analogue production.

A significant breakthrough in replicating meat flavour (and colour) in alternative products is the application of leghemoglobin, a naturally occurring symbiotic protein molecule synthesised in legume and many other plants (Singh and Varma 2017). A recombinant soybean leghemoglobin has been used by Impossible Foods for plant-based imitation hamburgers. The haem protein has a dual role of imparting meat flavour (blood) as well as meat-like colour. For processed meat, plant-derived spices and herbs are traditionally utilised to intensify the product’s overall aroma and taste; this strategy is also applied to alternative products using the same ingredients and similar formulations. However, it must be recognised that, due to the superior gelling and emulsifying capacity of myofibrillar proteins, protein-flavour interactions and volatile compound entrapments within processed meat protein matrixes can be very different from those of plant, algae, fungal, and insect protein-based products because of their generally lower functionality. On the contrary, the commodity-specific flavours (and off-flavours) that consumers are unfamiliar with or found objectionable tend to exacerbate the negative perception to developing meat alternatives.

For meat-like colour, a number of natural red pigments other than leghemoglobin are added to alternative products to mimic raw meat colour. Red beet extract, paprika, red rice extract, and carrot juice have been used in plant-based sausages and patties and mycoprotein-based hamburgers. In terms of thermostability, within the slightly acidic pH range (5.5–5.9) of fresh post-mortem meat, soy leghemoglobin was reported to denature between 70.2 and 78.6 °C (Tzoneva and Mishonova-Alexova 1998). Therefore, as an indicator of cooking doneness and microbiological safety, the loss of red colour due to denaturation of the pigment if used in alternative products could be a valuable quality and safety index. However, the colour change associated with end-of-cooking temperature is not expected for other organic colourants. Moreover, whether these small pigments are chemically reactive with nitric oxide (NO) in nitrite-cured products is not understood.

Nutrition and health

Meeting the overall nutritional standard of meat and that of muscle proteins in particular is also a challenge in developing meat analogues from alternative sources. Animal source proteins provide a complete essential amino acids profile suitable for humans. However, this is generally not true for nonmuscle proteins. For example, globular proteins from legume pulses are deficient in methionine; fibrinous cereal proteins used to mimic muscle-like fibrils are low in lysine. Therefore, to meet the nutritional requirement and reach the equivalent essential amino acids density, significantly higher amounts of plant-based meat analogues (i.e. more protein) would have to be consumed, leading to an extra caloric intake and potential uric acid stress (Fussell et al. 2021). In addition to proteins, edible plant materials are generally deficient in several vitamins and essential trace minerals that are naturally present in meat, for example, vitamin B₁₂, creatine, taurine, carnosine, as well as haem iron, zinc, and iodine (Neufingerl and Eilander 2021).

The health risk associated with meat and alternative products consumption is rather controversial and has been a subject of considerable debate. Meat (mainly beef, pork, lamb, and poultry) and meat products are perceived unhealthy by many consumers due to the high saturated fat and cholesterol content and misconceptions that meat contains residual antibiotics, growth promoters, and possible infectious vectors. Such risks are generally low due to strict regulations and monitoring programs for compliance during the course of meat production, e.g. zero tolerance for chemical and biological hazards before the animal is harvested. Wang et al. (2023) conducted a systematic literature review of randomised controlled trials on meat consumption and the health implications, and they found no relationship between meat intake and human gut health based on the available studies. Meanwhile, the safety of meat alternatives is grossly neglected and lacks controlled studies so far. For example, the ultra-high processing temperatures for plant protein fibrillisation could promote toxin formation (advanced glycation, lipid oxidation end products, etc.) and certain functional additives (e.g. carboxymethylcellulose and carrageenan) can induce intestinal inflammation (Martino et al. 2017). There has been paucity in literature addressing these health factors as well as other potential hazards.

On the other hand, limited yet encouraging studies show that the consumption of plant-based alternatives could promote gut health by the modulation of the microbial profile. Toribio-Mateas et al. (2021) reported positive changes in the gut microbiome of flexitarian consumers consuming plant-based meat alternatives as a part of their diet, claiming an increased butyrate metabolising potential and a decreased Tenericutes phylum population. Such information is scant yet highly relevant and significant; further research is required to gain a clear understanding of the molecular mechanism and the role of different chemical components present in the products in addition to proteins.

When edible insects are used for alternative products, the nutritional quality varies widely depending on the species and metamorphosis state (e.g. larvae, pupae, and adults). As comprehensively reviewed by Kouřimská and Adámková (2016), some species of edible insects contain good amounts of minerals as well as vitamins that are abundant in meat (e.g. Zn, Fe, and B-vitamins). Compared to plant protein, proteins from many insect species have the nutritional benefit relative not only to the total protein content but also to the level of essential amino acids (e.g. lysine, tryptophan, and threonine) and their bioavailability (Chen et al. 2010). Likewise, the nutritional value of microalgae varies between varieties and the environments in which they are grown. Proteins in edible algae species are generally of limited lysine and tryptophan content (Dawczynski et al. 2007).

To overcome nutritional differences, food processors and nutritionists would incorporate nutrients as additives in the product formation, and the ultra-formulation approach has raised health concerns. Most of the added nutrients are conceivably preserved in moderately cooked products; however, for extruded meat analogues, the high temperature, humidity, and pressure conditions would dispose added nutrients to thermal destruction unless they are added post-processing. Palanisamy, Töpfl, et al. (2019) indicated that the nutritional value of extruded lupin protein meat analogues could be improved by the fortification with polyphenol-rich spirulina. The chemical and microbiological safety of such products has not been carefully examined, and more research is required to close the knowledge gap.

For many insect-based protein products, a common food safety concern is the natural presence of bioactive toxins and allergen compounds (Kouřimská and Adámková 2016). Moreover, hazardous pesticide residues and heavy metals from the ecosystem could contribute to health risk of whole or parts of insects when used as alternative source of proteins. Therefore, as good as a potential alternative source of nutritious protein, insect-based products are subject to regular food safety evaluation and risk assessment (EFSA 2021). For microalgae protein, the main food safety concern is allergens. Mycotoxins could be produced during algae production and poses a health hazard, depending on the carbon source for production (Brzonkalik et al. 2011; Van der Spiegel et al. 2013). Hence, it is important to select the microalgae species that may be resistant to pathogens.

Conclusion and reflective comments

Eating less meat is increasingly considered a healthier dietary pattern and lifestyle, but this notion is not consistently supported by scientific data and often contradicts the evidence of overwhelming nutritional benefits of meat. On the other hand, reducing meat consumption is understandably a legitimate ethical option and viable approach to ensuring a sustainable protein food supply. Although there has been much debate whether traditional animal meat production should be gradually reduced and eventually replaced by alternative protein materials in the near future, the reality does not indicate or suggest a diminishing meat market or consumer demand for meat. It is almost certain that with the world population continuing to grow, meat production will correspondingly increase albeit at a likely slower rate. It is important to stress that with the constraint of meat supply, there would be a stronger demand for plant, microalgae, fungal, and other alternative proteins. This would underscore the complementary relationship of the expanding alternative protein market with traditional meat and meat products for nutrition requirement and eating experience.

As consumers’ curiosity begins to fade and major technological challenges go unresolved, the meat alternative market that once saw an exponential growth, particularly in the past few years, now seems to be settling and undergoing adjustments. This is evidenced by the stagnant growth or downsizing of some of the major plant-based businesses in the last year. To be successful, the strategy of developing meat alternatives entails a whole-system approach regardless of the source materials – plants, microalgae, single cells, or insects. Because of the inherent structural characteristics and amino acid composition of nonmuscle proteins, which differ sharply from the myofilamental structural system that imparts crucial tenderness, mouthfeel, and functionality of meat, it is not technologically possible to create or reconstruct myofiber analogues that can directly substitute or replace meat and meat products. Even if the fibril restructuring technology advances to the point at which muscle myofibrils can be closely mimicked, the potential damage to proteins and other nutrients due to over-processing would likely be a constraining factor for such endeavours.

Consistent with the slow technological advance and innovation, sales of refrigerated plant-based sausages and burgers in the United States in 2022 decreased compared to 2021 (Buss 2023). Yet, emulsified meat analogues (e.g. frankfurters and bolognas) experienced increased consumption during this period, suggesting greater opportunities in this sector where fibre structure is not a major prerequisite for product quality attributes. Aside from hyper-processing to form meat-like structure, the ultra-formulation strategy for establishing meat flavours (aroma and taste) and nutritive values subjects finished alternative products to safety concerns and potential liability. Such challenges are increasingly being examined in epidemiological studies and gradually brought to the public attention (Srour et al. 2022).

It should be soberly cognisant that, despite all the effort, what meat analogues provide, in a realistic sense, is a different eating and quality experience from animal meat and meat products. Therefore, it seems advisable to treat them as a different group of protein products to avoid the psychological dilemma and often, consumer disappointment and unrealistic expectation. As such, non-meat protein products should be considered as novel protein foods with their own identity and recognised only as alternatives to meat rather than analogues or substitutes. This approach would allow food innovators to focus on the sensory quality, nutrition, and safety of novel protein foods and not to unproductively simulate a benchmark food sector (meat) that is very difficult and economically challenging to replicate. Another interesting emerging idea is to develop hybrid meat products whereby a portion of meat is replaced by plant or other alternative source protein ingredients (Grasso and Goksen 2023). Such formulated meat products have been launched in Denmark, UK, Austria, and US markets (burgers, sausages, burgers, meatballs, etc.). Though not completely simulating meat, such formulated hybrid blends could still provide the familiar sensory attributes of meat (taste and texture) and muscle-specific nutrients (Baune et al. 2023). Finally, innovations that focus on improving the production efficiency and volume of protein crops, which are well-accepted and remain a primary source of alternative proteins, instead of pursuing exotic protein species, should be a consideration.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The original data of the paper are available upon request.

Additional information

Funding

The work is funded by USDA National Institute of Food and Agriculture, USA (Hatch Project 1020736).