Deep dive: Fermentation protein ingredients and food functionality

Overview of fermentation-derived protein ingredients

By 2050, the global population is projected to reach 9.7 billion, significantly increasing demand for essential macronutrients, particularly protein. Protein is vital for maintaining physiological functions and metabolic balance in the human body, and its deficiency can lead to hormonal imbalances and inefficient energy metabolism. (B. Wang et al. 2023)

The growing challenges and impact of our current agricultural system have increased interest in alternative protein sources for food (Poore and Nemechek 2019). Fermentation-derived (FD) alternative proteins (AP), typically referred to as mycoprotein or single-cell proteins (SCP), are a promising solution to global protein shortages, offering significant environmental and nutritional advantages. FD AP production offers notable advantages over conventional protein sources, including shorter production cycles, reduced land requirements, and suitability for diverse climates. New advances in food science and production are demonstrating the benefits of FD proteins, such as high protein, vitamin, and lipid content, and overcoming historical challenges in protein ingredient functionality, high nucleic acid content, low digestibility, and limited availability (Ugbogu and Ugbogu 2016). Progress over the past decade has led to considerable advances in the compositional and functional attributes of fermentation-derived alternative proteins.

Research and development in alternative proteins have driven rapid innovation. These products are increasingly appealing to mainstream consumers, particularly flexitarians. Over the past decade, the food industry has seen evolving demand for diverse protein sources. The development of alternative proteins through microbial fermentation has introduced two emerging industries in food production: precision fermentation (PF), which uses microbes to produce specific animal proteins, and fermentation to produce high-protein microbial biomass. Biomass proteins are classified as single-cell protein (SCP) when derived from yeast/bacteria/microalgae. Protein ingredients produced by filamentous or mycelial fungi are often called mycoprotein. These innovations in food technology and agriculture continue to significantly widen their market presence and organoleptic appeal (Hefferon et al. 2023). 

Fermentation has been used since ancient times for food preservation and has evolved into a diverse process for producing various fermented foods and beverages from many feedstuffs. Currently, three types of fermentation are available for food applications:

• Traditional fermentation produces foods and beverages using microorganisms like bacteria and yeasts, which may be naturally present or added as starter cultures. This process enhances the sensory and nutritional properties of food products by modifying their organoleptic qualities. While interest in traditional fermentation is resurgent, this deep dive will not extensively cover the functionality of food ingredients related to traditional fermentation.
• Biomass fermentation focuses on producing mycoprotein and SCP as high-protein ingredients that also provide other nutrients. Sometimes, biomass fermentation approaches use sidestreams such as food processing by-products and agricultural residues as substrates/feedstocks, an environmentally friendly and efficient approach for sustainable food production.
• Precision fermentation uses microbes to produce specific high-value molecules, such as proteins, pigments, vitamins, and fats. These molecules are biomanufactured and prepared as ingredients in food products like alternative meats and dairy. (Boukid et al. 2023).

Biomass-derived (BD) fermentation products have been commercially available for several decades, providing protein ingredients for center-of-plate products and other applications where functional proteins enhance end products. Precision fermentation (PF) for food ingredients represents a recent technological breakthrough, offering modeled resource efficiencies superior to traditional livestock farming (Knychala et al. 2024). Despite its seemingly recent application to food, PF approaches have been used for decades to produce food preparation enzymes like rennet and dough conditioners. With its potential to revolutionize food systems, PF focuses on producing milk proteins, egg proteins, and specific components for alternative meat products, enabling more sustainable and potentially localized food production (Nielsen, Meyer, and Arnau 2024).

Despite these advancements, we must address critical research gaps and challenges to realize the full potential of FD proteins in food applications. Key areas requiring attention include:

• Optimizing production efficiency to reduce costs and scale manufacturing for global markets.
• Enhancing sensory and functional properties, such as taste, texture, and aroma, to increase consumer acceptance of FD proteins.
• Navigating complex regulatory frameworks to ensure food safety and quality while fostering public trust.

In particular, understanding the functional behaviors of FD proteins — such as solubility (the ability to dissolve in water), emulsification (stabilizing oil-water mixtures), foaming (forming and stabilizing air bubbles), gelation (creating protein networks for texture), and water-holding capacity (retaining moisture for juiciness and texture) — is critical for replicating the qualities of animal-derived proteins in alternative products. Sensory properties, on the other hand, encompass the attributes perceived by the senses, such as taste (basic flavors like sweet, salty, sour, and bitter), texture (mouthfeel like hardness, cohesiveness, springiness, gumminess, and chewiness), aroma (smell that influences flavor perception), and appearance (visual elements like color and sheen). Enhancing these properties in alternative proteins is crucial to replicating the organoleptic appeal and functionality of animal-derived products, thereby improving consumer acceptance and overcoming barriers to adoption. Furthermore, these efforts will be pivotal for producing appealing dairy, egg, and meat substitutes. To meet consumer expectations, future innovations must focus on production methods, sensory attributes, and functionality to enhance these alternatives to broader markets.

This review provides a comprehensive overview of FD proteins for food applications, focusing on their microbial hosts, innovations, challenges, and opportunities in alternative meat, egg, and dairy production. Commercial innovation in FD APs began in the 1980s with BD products, whereas the commercialization of PF protein ingredients occurred far more recently. Therefore, much of the information summarized and reported here focuses on BD ingredients and their functionality.

Fermentation-derived ingredients and end products

FD proteins can be sourced from diverse microbes and similarly be used in a variety of applications, from meat, dairy, and egg substitutes to nutritional supplements, preservatives, colorants, texture regulators, and enzymes (Figure 3). This section highlights innovations in FD proteins enabling meat, egg, and dairy alternatives, exploring their applications, processes, and formulations.

Figure 3. The diverse roles of fermentation-derived proteins and molecules in food functionality. Created with https://BioRender.com.

Fermentation-derived meat protein ingredients and products

Alternative meat products are primarily categorized into two main groups based on their structure:

• Whole cut products made of fibrils (Figure 4). These products are designed to replicate the fibrous structure and texture of whole cuts of meat, such as chicken breasts or steak. 
• Restructured meat products (Figure 4), including coarse-particle products like ground/mincemeat and fine-particle (emulsion-based) products, such as gelled emulsions mimicking the smooth texture of hot dogs/frankfurters and deli meats. In fine-particle alternatives, achieving desired texture relies on gelation and forming a fine protein matrix, which eliminates the need for protein fibrillization (Xiong 2023).

To date, most examples of fermentation-derived biomass protein have come from fungi, thus more details are available regarding their applications and functionality as compared to microalgae and bacteria biomass protein ingredients. Commercialization of ingredients like mycoprotein began in the 1980s. These filamentous fungi-derived ingredients, along with other BD proteins, exhibit excellent gelling, emulsifying, and foaming properties, making them ideal for meat alternatives. Their filamentous structure and high protein content allow them to effectively mimic the textural properties of animal-derived proteins. Current fungal-based products include nuggets, burgers, fish fillets, and deli meats (Xiong 2023). Textured FD meat products can be developed using two primary approaches:

• Direct utilization of fungal biomass, producing microbial fermented meat. 
• Processing isolated fungal proteins to simulate meat structure.

The production of fungal-based meats often involves combining mycoprotein with gel-forming agents such as acacia bean gum, agar, and alginate, and binding the aggregates using egg white protein or plant-based binders. Some biomass may contain endogenous molecules like polysaccharides that contribute to gelling without exogenous agents. These mixtures are heated to induce crosslinking, followed by cooling and freezing, which helps control ice crystal growth. This process binds filamentous mycelium into fibrous bundles, mimicking the texture of meat with a “fleshy” structure. 

Modern techniques such as electrospinning, wet spinning, and 3D printing allow protein structuring at a molecular level, mimicking the intricate structure of animal meat (Figure 1). Additionally, larger-scale methods such as extrusion, freezing, shear cell technology, and hydrocolloid blending enhance the texture of fungal-based meat products by replicating animal-derived meat’s physical and structural properties. The combination of these techniques improves the overall quality and consumer appeal of meats (Figure 1; Figure 4) (B. Wang et al. 2023).

Figure 4. Production of meat alternatives from non-muscle proteins can be categorized into three types: 1: whole muscle-like structures, 2: coarse-particle products, and 3: fine-particle emulsion-based products (Xiong 2023). Created with https://BioRender.com.

Fungal biomass commercial end-products

The inherent structure of filamentous and mycelial fungal biomass has opened new avenues to its use in texturization and recreation of familiar end-products. For instance, combining Neurospora intermedia mycoprotein with plant-based excipients like soybean protein isolate and soluble starch results in well-textured and appealing end-products. Under optimized pH and salt concentrations, plus the addition of a soluble starch solution at 3%, these formulations balance elasticity, hardness, water retention, and fracture elongation. These organoleptic attributes are all key elements of texture acceptance. These findings demonstrate N. intermedia‘s potential for adoption as an AP meat ingredient (B. Wang et al. 2024). 

Recent advancements in fungal strains and production technologies have led to the development of diverse fungal-based meat alternatives. Fermentation-derived protein producers leverage strains like Aspergillus sp., F. venenatum, and Pleurotus eryngii to create diverse commercially available products, including chicken substitutes, seafood, and bacon alternatives. As the industry grows, there is an expectation that new, food-safe microbes will be utilized to produce BD ingredients.

Processing and formulation considerations for mycoprotein products

Mycoprotein has long been studied as a fermentation-derived biomass protein ingredient with potential for widespread commercial application. Work related to formulation and ingredient processing is of particular interest, especially in how it affects ingredient functionality in alternative meat products.

Processing to remove RNA must balance food safety with preserving product appeal. Thermal treatments to lower RNA content in microbial cells can partially denature proteins, affecting their functionality, while contributing to protein loss during ingredient preparation (Figure 1A). Enzymatic alternatives, such as the nuclease enzyme treatment, could effectively reduce RNA and DNA content without high heat exposure, thus preserving protein quality and concentration (US Food and Drug Administration, 2024). This PF-derived nuclease enzyme is an example of using a PF output as a processing aid for biomass fermentation-derived protein. Such enzymatic methods could offer an additional approach for the preparation and processing of BD protein ingredients. 

In one study, replacing chicken breast meat with mycoprotein in breaded nuggets demonstrated texture properties (hardness, springiness, cohesiveness, or chewiness) comparable to conventional chicken nuggets. These nuggets exhibited lower lightness and yellowness color values but showed reduced cooking loss, ultimately achieving acceptable sensory and technological qualities (Hashempour-Baltork et al. 2023).

A key organoleptic benefit of mycoprotein is its ability to form textured, fibrous structures that resemble animal-derived meat products. However, binding is still required for final formulation into end products with the expected texture and mouthfeel. Typically, 3% egg white protein (EWP), primarily ovalbumin, is added to enhance gelation through disulfide bridges and improve the texture. A recent study explored mycoprotein (MYC)-EWP interactions, demonstrating that mycoprotein exhibited viscoelastic properties that were influenced by pH, salt type, and concentration. Acidic conditions increased storage modulus (G′), while calcium chloride (CaCl₂) had a greater impact than sodium chloride (NaCl) due to calcium crosslinking (Okeudo-Cogan et al. 2023). Imaging confirmed that EWP coated the fungal hyphae completely, moderating these effects. However, for price stability, allergenicity, and sustainability, it would be beneficial to identify alternatives to whole chicken EWP.

Studies exploring alternatives to EWP, such as potato protein (PoP) and multivalent cations (e.g., Fe³⁺ and Ca²⁺), revealed significant improvements in gel strength, texture, and nutritional quality (Okeudo-Cogan et al. 2024). Fe³⁺ influences fungal hyphae aggregation, particularly at lower pH levels, where it enhances protein-protein interactions but reduces composite strength by disrupting inter-hyphal binding. Conversely, CaCl₂ improves gel strength and texture, highlighting the dominant structuring role for calcium ions. When used together, Fe³⁺ and Ca²⁺ synergistically reduce excessive protein aggregation and enhance the overall structural stability of MYC-PoP composites. Interestingly, the effects of Fe³⁺ are pH-dependent, with acidic conditions promoting aggregation and weaker inter-hyphal interactions (Figure 6C). Therefore, these findings highlight the critical role of pH, ionic strength, and additive interactions in balancing protein aggregation and structural integrity. Moreover, this study demonstrates that PoP enhances fungal-protein composites by mediating electrostatic interactions, particularly in the presence of salts, positioning it as a promising allergen-free alternative to EWP (Okeudo-Cogan et al. 2024).

In addition to alternative chicken products, mycoprotein has shown remarkable potential in replacing traditional ingredients in sausages, nuggets, and patties. Studies replacing meat with mycoprotein in sausage formulations improved the nutritional profile of sausages by increasing essential amino acids (EAAs) and unsaturated fatty acids (UFAs). The closed nature of fermentation and the controlled environment in which the ingredient is processed may also reduce food contamination risks and increase microbial safety compared to meat sausages. Additionally, these sausages demonstrated superior water- and oil-binding capacities with potential for texture optimization (Shahbazpour et al. 2021).

Indeed, FD mycoproteins are a promising base ingredient for alternative meat formulations. Their potential for traditional center-of-plate product texture attributes, water holding capacity, and versatility makes them a large share of the biomass protein ingredients offered. However, the research space to find appropriate binders that can be used across mycoprotein products and the reduction of flavoring ingredients in some products (like the vegan burger, Table 2) highlight near-future food science and formulation advances that can further increase mycoprotein versatility and appeal.

Fermentation-derived alternative dairy ingredients

Alternative dairy products will become increasingly essential for sustainable and ethical food production. The growing demand for alternatives to livestock-derived dairy products has led to innovations in producing dairy-like proteins derived from fermentation and plant-based sources. A key challenge lies in replicating the nutritional profile, texture, and functional properties of dairy proteins, such as caseins and whey, while ensuring environmental sustainability and catering to the preferences of health-conscious and ethical consumers.

Milk contains approximately 87–89% water, with the remaining fraction consisting of proteins (3.0–3.9%), fats (3.3–5.4%), and sugars (4.4–5.6%), and trace amounts of salts and vitamins. The protein fraction consists of 80% caseins and 20% whey proteins, making it highly nutritious due to its complete essential amino acid profile. Whey proteins, including β-lactoglobulin (β-LG, 52%), α-lactalbumin (α-LA, 17%), glycomacropeptides (12%), immunoglobulins (10%), serum albumin (5%), and lactoferrin (1.5%), are used in various applications such as infant formula, yogurt, cheese, and sports nutrition products. β-LG, a key bovine whey protein, is nutritionally valuable despite being absent in human milk. Caseins, including β-casein (36%), κ-casein (14%), α-s1-casein (40%), and α-s2-casein (10%), are heat-stable proteins with unique functional properties such as emulsification and water-binding, making them particularly suitable for coffee whiteners and other heated products (Nielsen, Meyer, and Arnau 2024).

The recombinant production of casein and whey proteins has made remarkable strides through microbial fermentation. Microbial hosts like K. phaffii, S. cerevisiae, and E. coli produce recombinant bovine κ-casein and other dairy proteins, with significant progress achieved in the yield and efficiency of recombinant protein production (Eastham and Leman 2024). For instance, companies are utilizing PF to scale up to commercial production of bovine β-casein for animal-free cheese alternatives, replicating the texture, flavor, and melting profile of mozzarella. Similarly, whey proteins like α-lactalbumin and β-lactoglobulin are efficiently produced in microbial systems, enabling sustainable dairy alternatives (Iglesias-Figueroa et al. 2016; Kim et al. 2007).

Recombinant dairy proteins, such as β-lactoglobulin and α-lactalbumin, are increasingly being used in the dairy industry due to their excellent functional properties, including emulsification, foaming, and heat stability (Diaz-Bustamante et al. 2023; Hoppenreijs et al. 2024). FD production and subsequent isolation of recombinant dairy proteins produce a homogenous white powder suitable for various food applications (Diaz-Bustamante et al. 2023). However, these proteins are rarely used in isolation. They are marketed to food manufacturers, sold in bulk, and used as ingredients for various alternative dairy applications. Often, formulators integrate recombinant dairy proteins with plant-based fats, carbohydrates, salts, vitamins, calcium, and minerals to create cheese alternatives (Diaz-Bustamante et al. 2023). These proteins are produced through PF using microorganisms like Trichoderma reesei, Pichia pastoris, and K. lactis, which are capable of producing commercial titers (~15g/L and higher) of recombinant proteins with the required post-translational modifications (Diaz-Bustamante et al. 2023).

PF has become a key technology for producing recombinant milk proteins. Companies like Perfect Day employ Trichoderma reesei to produce β-lactoglobulin, the first commercial example of a PF whey protein for products such as ice cream and alternative milk products (Figure 7A). Other companies, including New Culture and Change Foods, focus on producing recombinant caseins using PF technologies (Nielsen, Meyer, and Arnau 2024). 

Whey proteins are proportionately the largest protein fraction of milk and contribute to many of the functional attributes of dairy products. In bovine milk, β-lactoglobulin is the major whey protein; therefore, there is a high interest in producing the protein using PF approaches.  At research scale, this protein was expressed from engineered T. reesei fungi, purified, and tested to demonstrate functionality comparable to its cow milk-derived counterpart (Aro et al. 2023). Bovine β-lactoglobulin exhibited exceptional emulsification properties, forming stable emulsions without phase separation for 30 days at 4°C, making it ideal for long-shelf-life food products (Figure 7B).

While recombinant whey production has seen earlier development and commercialization, various companies and startups are now turning to casein. Progress on PF-derived caseins has been slower, largely due to their more complex interactions with calcium salts and fats (Diaz-Bustamante et al. 2023). 

Nevertheless, the production of casein proteins through biotechnology also holds great potential. Reconstituting casein protein with calcium and fats produces the micelle structures that agglomerate into cheese curds, a process that is crucial for traditional cheese-making. To date, the ideal peptide length, post-translational modifications, and downstream processing pathways to create a PF casein ingredient, and subsequent micelle formation, are still under investigation (Chezan et al 2026). For example, the phosphorylation of casein appears to play a role in its interaction with fat and calcium, and fully dephosphorylated recombinant casein fails to properly fall out of solution and interact with calcium (Che et al. 2025). Indeed, recombinant phosphomimetic casein protein, where serine amino acids are replaced by aspartic acid in the protein sequence, were able to achieve casein micelle formation, paving the way for additional avenues towards casein functionality and micelle formation (Balasubramanian et al. 2025). 

Attempting to recreate the exact biochemistry of cheese curds may not be required for successful PF-casein containing cheese formulations. Recently, patent filings have disclosed non-micelle-based methods for achieving casein functionality in alternative cheeses (Radman 2023). Using these biotechnologically produced caseins, PF-enabled cheese typically requires blending PF-derived casein with water, oils, and small quantities of carbohydrates. Emulsifiers, stabilizers, and/or flavoring agents are also potential ingredients, depending on the type of cheese produced. The mixture is subjected to heating and mechanical processing to achieve a texture, meltability, and stretchability similar to traditional cheese (Figure 7C). The final product is shaped into various forms, such as blocks, slices, or shreds with additional treatments to improve its sensory properties. These cheese analogues exhibit a smooth and cohesive texture, excellent melting and stretchability, and a customizable flavor profile that closely mimics traditional dairy cheese. Casein proteins contribute essential functionality to cheese with the ability to bind to fats (melting), induce increased binding when cooked (stretching), and ionic attraction of salts (taste). This innovative approach provides a sustainable and versatile alternative to traditional cheese production (Radman 2023). 

An example innovation in plant-based dairy alternatives is the implementation of concentrated microalgae flour to produce lactose-free milk with a nutritional profile comparable to cow’s milk, including high-quality essential amino acids (Wu et al. 2023). Unlike previous approaches that used algae as supplements or additives, current efforts focus on a fully algae-based dairy substitute derived from protein-rich microalgal flour (Cornall 2021; Ho 2021). By adjusting the ratio of water-soluble microalgae flour, formulators can customize protein content and enhance sensory properties, paving the way for a range of alternative dairy products like yogurts, cheeses, and ice creams.Ice cream is an example of a commercial application opportunity for fermentation-derived protein ingredients. An FD protein ingredient with plant-based fats and sugars can form the basis of an animal-free ice cream. In some cases, these proteins can be biomass-derived, such as in the case of the collaboration between the Danish Technological Institute and Sophie’s Bionutrients, which developed a Chlorella microalgae-based ice cream with a higher protein content than many plant-based alternative ice creams (Figure 7D) (Southey 2022). Similarly, PF-derived beta-lactoglobulin protein adds protein content and creamy texture to ice cream (Smith 2024). In both examples, FD protein ingredients drive organoleptic improvement of animal dairy-free desserts while enabling lactose-free formulations.

Figure 7. A) A whipped alternative dairy product with PF β-lactoglobulin to enhance foaming  (photo courtesy of Perfect Day, used with permission); B) Left: Glass vial of PF-derived bovine β-lactoglobulin and right: Glass vial of control animal-derived β-lactoglobulin were stored undisturbed at 4 °C for 30 days (Aro et al. 2023 under the terms of the CC BY license); C) Mozzarella cheese using PF casein protein demonstrating stretching and melting (photo courtesy of New Culture, used with permission); D) An ice cream product using microalgae-derived protein as an ingredient, demonstrating creaminess in an animal dairy-free formulation (photo courtesy of Sophie’s Bionutrients, used with permission); E) A whipped egg ingredient using PF ovalbumin demonstrating foaming (Bioalbumen®, photo courtesy of Onego Bio, used with permission); F) A bread baked using PF ovalbumin demonstrating binding in baked goods (Bioalbumen®, photo courtesy of Onego Bio, used with permission). Created with https://BioRender.com.

Fermentation-derived alternative egg protein ingredients and products

PF offers a promising approach to replicate the functional and nutritional properties of eggs without animal-derived components. Raw eggs consist of 12.5–13% protein, with ovalbumin comprising the majority (54%) of egg white proteins. Other key proteins include ovotransferrin (12%), ovomucoid (11%), and smaller amounts of ovomucin and lysozyme (each 3.5%). These proteins exhibit desirable functional properties, such as foaming, emulsifying, and gelling, making them invaluable in cheese production, confectionery, and baking (Nielsen, Meyer, and Arnau 2024).

Egg whites also contain biologically active proteins with various functional benefits. Lysozyme, for example, is used as a food preservative due to its antimicrobial properties, ovotransferrin functions as a metal transport agent with antimicrobial and anticancer effects, and ovomucin and ovomucoid exhibit tumor-suppressive properties. These qualities emphasize the importance of eggs in global diets and industrial applications (Knychala et al. 2024).

Egg ingredient alternatives have gained significant attention due to animal welfare, sustainability, and global food security concerns. Eggs are valued for their high nutritional content and their functional properties, such as coagulating, emulsifying, foaming, and coloring. However, the contribution of animal-based foods to global carbon emissions, poultry diseases reducing egg supply, and unstable pricing have driven consumer and industry interest in egg alternatives. As a result, the global egg alternative ingredient market has a projected 2025 value of $1.6 billion US dollars (Jin, Seo, and Kim 2024).

PF egg white proteins offer several advantages, including scalability, reproducibility, and absence of contaminants such as pathogens commonly associated with egg proteins. In isolation, it also enables the production of specific egg white proteins, such as ovalbumin, lysozyme, and ovotransferrin, enhancing their functionality for targeted applications. The PF-derived proteins closely replicate the functional properties of egg proteins across various food applications. Patent filings demonstrate various functional claims for PF-derived ovalbumin (Mahadevan et al. 2021).  For example, PF-derived ovalbumin provides excellent foaming and emulsification, ensuring light textures in baked goods (Figure 7E). The foaming properties are especially useful in products like cakes and meringues, where stable foam structures are essential for texture and volume (Figure 7F). Recombinant ovalbumin stabilizes and emulsifies in mayonnaise and dressings, maintaining smooth textures and sensory qualities in animal-free formulations. It also enhances binding and gelation in plant-based meat alternatives, improving texture and mouthfeel to mimic animal-based counterparts. Fine-tuning ovalbumin’s gelling properties through temperature, pH, and salt can create specific textures in alternative meats, offering versatility in formulation. Ovalbumin’s gelling ensures stability, creaminess, and a premium texture in dairy-free desserts like mousses and ice creams. Finally, recombinant ovalbumin is a high-quality protein in nutritional supplements and protein powders, offering allergen-free, clean-label solutions for health-conscious consumers (Mahadevan et al. 2021). 

Recent advancements in PF have enabled the production of ovalbumin and other egg proteins in microorganisms (Nielsen, Meyer, and Arnau 2024). Recombinant production of egg white proteins, particularly ovalbumin, has been explored since the 1970s using hosts such as E. coli, S. cerevisiae, and K. phaffii. While initial attempts yielded minimal production, recent advancements with hosts like K. phaffii have enabled production levels of 5.45 g/L for ovalbumin, 0.1 g/L for chicken ovotransferrin, and smaller proteins like lysozyme (0.4 g/L) and avidin (0.33 g/L) (Knychala et al. 2024). Recent commercial breakthroughs include the production of ovalbumin in Trichoderma reesei, in Aspergillus niger, and Komagataella phaffii (Nielsen, Meyer, and Arnau 2024). Companies have made strides in producing EWP at high titers, with Every Company reporting ovalbumin titers from 17-30 g/L in K. phaffii and Onego-Bio reporting 120 g/L in T. reesei, which are significant strides compared to the literature published titers in S. cerevisiae (Jin, Seo, and Kim 2024), K. phaffii (Yang et al. 2009), andT. reesei (Aro et al. 2023) for EWPs. These developments highlight progress on commercial scalability and reproducibility of PF for egg protein production, signaling their near-term availability for commercial food production.

Further, these proteins provide functionalities higher than or equivalent to animal-derived egg whites (Knychala et al. 2024). Patents in this area emphasize producing innovative recombinant egg-white protein compositions. For example, methods for expressing two or more egg-white proteins through PF and producing recombinant ovalbumin for egg-free food formulations have been filed for IP protection (Anchel 2016; Mahadevan et al. 2021). PF companies have patent-protected innovative solutions on highly soluble egg-white proteins and egg white proteins from other bird egg species with enhanced functionality, like gelation over traditional hen egg ovalbumin.

Food safety and regulatory considerations of fermentation-derived ingredients

The global growth of the alternative protein sector depends on establishing clear, consistent, and harmonized regulatory frameworks. Such frameworks ensure consumer safety and facilitate international trade and market access. This section examines the regulatory landscape and safety challenges of FD proteins, emphasizing the importance of transparency and innovation in fostering consumer trust.

The global progress of alternative proteins in the food industry can be incremental due to the lack of harmonized national and international regulation frameworks that govern novel foods. Alternative proteins are typically categorized as novel foods, defined as foods or ingredients without a history of safe use, though regulatory approaches vary globally. Check out GFI APAC’s Novel Food Regulations Around the World website for a more detailed analysis. In the European Union, novel foods are regulated under the European Commission’s 2015/2283 legislation, effective from 2018. Conversely, the United States lacks a specific definition or regulation for novel foods. The U.S. Food and Drug Administration (FDA) treats new food ingredients as either food additives requiring premarket approval or to be evaluated and greenlit for food use as generally recognized as safe (GRAS). In Asia, regulatory frameworks for novel foods are less developed than in Europe or the U.S., with only a few countries, such as China, South Korea, Singapore, and Thailand, having specific regulations. China follows a model similar to that of the EU. At the same time, South Korea has temporary standards for novel ingredients, including recent provisions allowing plant-based meat alternatives to use the term “meat” on labels. Singapore and Thailand, however, have more comprehensive safety assessment guidelines. Singapore requires safety evaluations of novel ingredients under international standards, while Thailand mandates clinical or sub-chronic studies to ensure consumer safety. In Europe, the European Food Safety Authority (EFSA) evaluates the safety of novel foods, including PF-derived products, before issuing regulatory approvals. While EFSA has yet to approve PF protein food products, several are under review. Strains of microbial species like Bacillus subtilis, Saccharomyces cerevisiase, Chlamydomonas reinhardtii, and Corynebacterium glutamicum have a long history of safe use in food production (EFSA Panel on Biological Hazards). International organizations such as the Food and Agriculture Organization (FAO), World Health Organization (WHO), FDA, and EFSA recognize these strains as safe, making them suitable candidates as host microorganisms for PF applications (Mefleh and Darwish 2024).

Across jurisdictions, there are separate considerations for genetically modified organisms, which cover many of the host organisms used to produce precision FD ingredients, even if these organisms are not present in the final product (Hanlon and Sewalt 2020). These regulatory differences highlight the need for global cooperation and harmonized guidelines to facilitate the growth of the alternative protein industry (Malila et al. 2024).

PF-derived meat, dairy, and egg proteins are a rapidly growing food category. In the U.S., regulatory greenlighting of specific proteins, such as β-lactoglobulin (β-LG) from Perfect Day and ovomucoid from The EVERY Company, has led to their commercial sale and use as a food ingredient. One key issue is determining appropriate usage levels in food products to ensure safety and functionality. Another challenge is enhancing consumer awareness of the ingredients produced by PF. If consumers are allergic to animal-derived dairy or egg proteins, it’s highly likely they will be allergic to the PF-derived replacement protein. Therefore, unambiguous identification and labeling of these novel ingredients is necessary to enhance consumer awareness of this equivalence and potential allergenicity. Harmonizing regulations and improving transparency are vital to increasing consumer confidence and supporting the broader adoption of PF proteins in the food industry (Nielsen, Meyer, and Arnau 2024).

The safety evaluation of PF products focuses on the potential toxicity and allergenicity of the host microbe, its accompanying (copurifying) molecules, and the food ingredient itself. PF-derived proteins may introduce new allergens or alter the allergenic properties of the product due to changes in protein sequence. Comprehensive safety testing is required to mitigate these risks, including analyzing amino acid sequences for similarities to known allergens (Goto et al. 2023; Grundy et al. 2024). For instance, the FDA approved yeast-produced β-lactoglobulin for use in plant-based foods (excluding infant milk) only after rigorous safety assessments, including scrutinizing the product for potential allergens (US Food and Drug Administration, 2020). In biomass fermentation, rare adverse reactions to mycoprotein products have been reported in individuals with mold allergies (Katona and Kaminski 2003). Therefore, mycoprotein products carry labels with precise wording describing the product as potentially allergenic for those with mold allergies.

The allergenicity risks associated with PF technology remain a potential concern due to an emerging understanding of the metabolites produced by modified microbes. Possible risks include the production of known allergens, the modification of proteins to become allergenic, or the creation of novel compounds with unknown allergenic properties. For example, thermal processing can inactivate many heat-labile allergens, while others retain their allergenicity even after heat treatment. High temperature processing can also lead to the formation of potentially hazardous compounds, such as those produced by the Maillard reaction during dry heating, which may create new allergens. Further research is needed to understand how thermal processing impacts the allergenicity and safety of novel proteins.

The potential production of toxic metabolites by microbes and contamination with undesirable microorganisms are concerns that must be scrutinized and derisked during product development. Mycotoxins are toxic substances produced by a subset of filamentous fungi under specific conditions, pose a potential hazard, and can accumulate in tissues. Many foods have a risk of mycotoxin accumulation due to fungal contamination, such as soil-grown tubers, legumes, grains, and dried fruit. Improper processing or storage can lead to mycotoxin exposure, which poses a high risk to sensitive populations such as pregnant people, children, geriatric populations, and immunocompromised individuals. Utilizing sidestreams or feedstocks where molds have propagated requires extra care and surveillance to avoid transferring mycotoxins to yeast biomass. Strict processing protocols and safety measures, employed during GMP (“good manufacturing practices”) food production, are essential to mitigate these risks (Jach et al. 2022). Therefore, these mycotoxins are routinely tested for, and demonstrating their absence is typically considered a regulatory hurdle that must be met. Additionally, regulators often require the genetic sequences of microbes to be analyzed and the identification of any known mycotoxin-producing genes to be declared. For example, while some strains of F. venenatum can produce mycotoxins, the strains used commercially by Quorn do not make these toxins (O’Donnell, Cigelnik, and Casper 2018). Continued scrutiny and surveillance of mycotoxins, common in the food industry, is also required in fermentation settings, especially when investigating novel microbes and production conditions (Malila et al. 2024).

International cooperation among regulatory agencies is crucial to advance the alternative protein sector. Establishing globally accepted safety and quality standards would reduce trade barriers and ensure a level playing field for producers. Research into advanced safety evaluation techniques, such as proteomic and metabolomic profiling, could enhance the identification of potential allergens or toxins, providing more comprehensive safety assessments. Public engagement and education are equally important for addressing consumer concerns regarding the safety, sustainability, and nutritional value of FD proteins. 

By fostering trust, harmonizing regulations, and supporting ongoing innovation, the alternative protein industry can overcome its current challenges and pave the way for widespread acceptance and adoption of these innovative food solutions.