Innovation in precision fermentation for food ingredients

Abstract

A transformation in our food production system is being enabled by the convergence of advances in genome-based technologies and traditional fermentation. Science at the intersection of synthetic biology, fermentation, downstream processing for product recovery, and food science is needed to support technology development for the production of fermentation-derived food ingredients. The business and markets for fermentation-derived ingredients, including policy and regulations are discussed. A patent landscape of fermentation for the production of alternative proteins, lipids and carbohydrates for the food industry is provided. The science relating to strain engineering, fermentation, downstream processing, and food ingredient functionality that underpins developments in precision fermentation for the production of proteins, fats and oligosaccharides is examined. The production of sustainably-produced precision fermentation-derived ingredients and their introduction into the market require a transdisciplinary approach with multistakeholder engagement. Successful innovation in fermentation-derived ingredients will help feed the world more sustainably.

Keywords:

• Fermentation
• synthetic biology
• food ingredients
• alternative proteins
• lipids
• oligosaccharides

Introduction

More sustainable food production methods and food process operations, which use less of the world’s natural resources, are needed to support a growing population and our food systems for food and nutrition security (Béné et al. 2019; FAO et al. 2021; Knorr and Augustin 2022). Traditionally, land was used for the production of plant and animal-based food. Plants and animals from the oceans and waterways were also harvested for food. The drive for more sustainable sources of food has resulted in increasing interest in the production of cellular products (e.g., in vitro meat) and the use of microbes as hosts for the production of acellular products (e.g., food ingredients) (Anderson, Islam, and Prather 2018; Rischer, Szilvay, and Oksman-Caldentey 2020; Teng et al. 2021). Edible biomass (e.g., algal biomass) is also becoming attractive as a source of sustainable food (Stengel and Connan 2015). Fermentation, which includes traditional fermentation, biomass fermentation, and precision fermentation, has an important role in building next-generation food ingredients and products (Good Food Institute 2021; Teng et al. 2021). Products made by fermentation have reduced reliance on land, lower greenhouse emissions, and use less water compared to traditional agriculture (Teng et al. 2021).

Traditional fermentation has been used for centuries as a means to preserve foods and increase the nutritional value of foods. Fish, meat, milk, vegetables, legumes, cereals, and fruits were used as the source material for fermentation for the purpose of preserving food. Fermented foods are produced through the controlled growth of microorganisms and conversion of components in the food through the action of microbial enzymes. Well-known fermented food and beverage products include yogurts, kimchi, kefir sauerkraut, cheese, tempeh, wine, beer, and natto (Bourdichon et al. 2021; Marco et al. 2017; Shiferaw Terefe and Augustin 2020). Microorganisms naturally present in the raw food can result in spontaneous fermentation (e.g., traditional production of sauerkraut, kimchi, and sourdough bread) or starter cultures may be added to the food to initiate fermentation to obtain a more standardized fermented product (e.g., Rhizopus oligoporus for tempeh, Bacillus subtilis natto for natto, Aspergillus oryzae for miso, kefir grains which contain yeasts such as Kluyveromyces marxianus, Saccharomyces cerevisiae, Saccharomyces unisporus in addition to lactic and acetic acid producing bacteria for kefir) (Dimidi et al. 2019).

In biomass fermentation, edible microorganisms (e.g., yeasts, bacteria, filamentous fungi or algae) are used as alternatives to conventional food sources produced by traditional agriculture (Bernaerts et al. 2018; Linder 2019; Ochsenreither et al. 2016; Ritala et al. 2017), both as a source of edible biomass and functional ingredients (polysaccharides, proteins, lipids/omega-3 fatty acids, vitamins, minerals, and dietary fibre) (Barros de Medeiros et al. 2022). Due to the nutritional composition, functional diversity and metabolic flexibility of algae, and in particular microalgae (e.g., Arthrospira platensis, Spirulina platensis, Chlorella vulgaris, and Dunaliella salina), they have been developed and cultivated as sources of proteins and other food components (Becker 2007; Garofalo et al. 2022). For example, microalgae are promising ingredients for the production of nutritious meat analogues, but there are still technical challenges in texturing the microalgae, the removal of undesirable odors and colors, and the scale up of cultivation and processing conditions (Fu et al. 2021).

Fermentation has also been used for the production of minor food components (e.g., antioxidants, colorants, flavors, enzymes, and vitamins). Recent exploration of extracts from freshwater algae (Spirogyra sp., Cosmarium sp., and Cosmarium blytii) indicated that they were high in carbohydrates, contained free amino acids (glutamic acid, aspartic acid and proline), and exhibited high antioxidant activity, making them potential sources of functional ingredients for food and feed (Santiago-Díaz et al. 2022). Increasingly, food waste is being used as the fermentation medium for the production of antioxidants, natural preservatives, colors, enzymes, and other food components. The utilization of an otherwise unused/under-utilized resource reduces food waste and also has the potential to create high-value products. Recently, single-cell protein has been produced by the fermentation of food waste by Yarrowia lipolytica (Yang et al. 2022). Fermentation can increase the extraction efficiency of compounds from the food matrix, modify the antioxidant profile, and create new bioactive compounds. Some examples of food waste fermentation include the production of (1) flavonoids and carotenoids from fruit or vegetable waste, (2) bioactive peptides from cereal processing waste and whey from cheese making, (3) antioxidative compounds from by-products of livestock and seafood processing (Martí-Quijal et al. 2021; 4) enzymes (glucoamylase) from a variety of substrates such as tea waste, rice bran, wheat bran, and corn stover (Wang et al. 2009), and (5) microbial red, orange, and yellow pigments from various food industry wastes as medium (e.g., whey, tomato waste, cottonseed meal, fruit seeds, etc.) (Panesar, Kaur, and Panesar 2015; Mehri et al. 2021).

Precision fermentation has been identified as an emerging food trend in the fourth industrial revolution of the food industry (Hassoun et al. 2022). Rapid advances in precision biology has enabled the programming of microbes to produce complex organic molecules (Tubb and Seba 2019). However, the commercialization of genetically modified organism (GMO) technology for foods has been a challenge due to public perceptions and knowledge of their safety, risks, labeling, and regulation (Burke 2012; Pappalardo, D’Amico, and Lusk 2021; Rzymski and Królczyk 2016). Alternative strategies for developing more functional non-GMO microbes through conventional non-GMO methods (Pérez-Torrado et al. 2015) has been explored because of the challenges in bringing GMO foods to market. Many of the early-entry fermentation products in the market have used non-genetically modified organisms in traditional and biomass fermentation platforms for the production of food ingredients. However, learning from the mistakes of the past associated with the introduction of GMO technology is essential for developing appropriate strategies to facilitate the introduction of GMO foods into the market.

The use of precision fermentation for the production of major food components (proteins, lipids, and carbohydrates) is emerging as an attractive option for the transformation of food systems. This trend is being driven by global needs to produce foods more sustainably. This paper discusses innovation in the food industry enabled by fermentation, with a focus on the developments in precision fermentation-derived food proteins, lipids and carbohydrates. It considers the market opportunities for fermentation-derived food ingredients and issues for bringing fermentation-derived food ingredients to market. The review covers aspects of business and markets for fermentation-derived ingredients, including policy and regulations. A patent landscape of fermentation for the production of major food ingredients (proteins, lipids and carbohydrates), is included as it forms part of the due diligence required for informing science strategies for fermentation businesses. The technological considerations for the development and exploitation of the precision fermentation value chain including the (1) choice of the feedstock for fermentation, (2) use of synthetic biology tools to tailor microbes for efficient production in the fermentation process, (3) downstream processing operations to recover the food component of interest, (4) scale-up of the fermentation process, and (5) characteristics of new fermentation-derived food products that are acceptable to consumers are reviewed. Figure 1 is a schematic which outlines the aspects that are covered in this review related to the innovation in fermentation derived food ingredients.

Figure 1. Considerations for bringing precision fermentation-derived ingredients to market.

Methods

Literature searches on publications relating to the various aspects on the science of fermentation and application in food systems (strain engineering, fermentation process and downstream processing, and functional performance of food ingredients) were carried out using the Web of Science. Patent landscape searching was done to assess the published patent literature. For obtaining information on business and market insights, the grey literature was also accessed. The searches were focused on the fermentation for the production of proteins, lipids, and carbohydrates (more specifically oligosaccharides).

Patent landscape search for fermentation-derived food ingredients

Broad patent landscape searching and analysis were conducted in relation to technologies for the use of precision fermentation for the production of food ingredients and alternative products such as milk, dairy, and meat, made from these food ingredients. More specifically, the search was conducted on the food ingredients of interest, namely proteins, lipids (fats/oils, triglycerides, phospholipids and glycolipids) and carbohydrates (polysaccharides, gums, oligosaccharides, and prebiotic dietary fibers). For the purpose of this search, analysis and reporting, precision fermentation is defined as the use of synthetic biology and specifically genetic engineering to insert specific genes into the DNA backbone of single-cell organisms and microorganisms to produce desired fermentation characteristics and products. The search was conducted in January 2022 and covers the time period from 2000–2021.

The search strategy used combinations of the following key words to locate relevant documents: “precision ferment+”, “cellular agricultur+”, “synthetic + biology+”, synbiolog+, syn_biolog+, synbio, syn_bio, microorganism+, micro_organism+, “micro + organism+”, single_cell_organism+, “singl + cell + organism+”, microb+, fungi, fungus, yeast+, ferment+, recombinant+, gene+, DNA, RNA, modif+, chang+, manipulat+, engineer+, transgenic+, “bio + engineer+”, bioengineer+, bio_engineer+, “bio + modif+”, biomodif+, bio_modif+, program+, protein+, fat, fats, lipid+, triglyceride+, tri_glyceride+, phospholipid+, glycolipid+, carbohydrate+, oligosaccharide+, “dietary fib+”, polysaccharide+, poly_saccharide+, gum, gums, food, foods, dairy, milk+, butter+, cheese+, meat and meats (where + indicates a word truncation, ““indicates a combination of words within a certain proximity, and _ represents a space or single character between two words). These key words were searched in all cases in the title, abstract and independent claims fields of patent documents, and extended to the full description in some cases. The search used the International Patent Classifications (IPCs) and/or Cooperative Patent Classifications (CPCs) listed in Table 1, and also included known entities of interest.

Table 1. Details of relevant International Patent Classifications (IPCs) and / or Cooperative Patent Classifications (CPCs) covered by the search strategy.

Classification	Details
A23 FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES	A23C DAIRY PRODUCTS, e.g., MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF … A23C9/15 • Reconstituted or recombined milk products containing neither non-milk fat nor non-milk proteins … A23C9/1512 • • {containing isolated milk or whey proteins, caseinates or cheese; Enrichment of milk products … A23C11/00 Milk substitutes … A23C11/02 • containing at least one non-milk component as source of fats or proteins … A23C11/08 • • containing caseinates but no other milk proteins nor milk fats A23C21/00 Whey; Whey preparations … A23D EDIBLE OILS OR FATS, e.g., MARGARINES, SHORTENINGS, COOKING OILS (animal feeding-stuffs A23K10/00-A23K20/30, A23K30/00-A23K50/90; foods or foodstuffs containing edible oils or fats A21D, A23C, A23G, A23L; obtaining, refining, preserving C11B, C11C; hydrogenation C11C3/12) A23G COCOA; COCOA PRODUCTS, e.g., CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF A23J PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS A23J1/00 Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites A23J3/00 Working-up of proteins for foodstuffs A23L FOODS, FOODSTUFFS, OR NONALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g., COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT … A23L2/00 Nonalcoholic beverages; Dry compositions or concentrates therefor; Their preparation (soup concentrates A23L23/10; preparation of nonalcoholic beverages by removal of alcohol {C12H3/00}) A23L2/52 • Adding ingredients (adding preservatives A23L2/44) A23L2/60 • • Sweeteners A23L2/66 • • Proteins A23L27/00 Spices; Flavoring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof A23L33/00 Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof A23L33/10 • using additives (addition of substantially indigestible substances A23L33/21) A23L33/115 • • Fatty acids or derivatives thereof; Fats or oils A23L33/12 • • • Fatty acids or derivatives thereof A23L33/125 • • containing carbohydrate sirups; containing sugars; containing sugar alcohols; containing starch hydrolysates (indigestible substances A23L33/21) A23L33/14 • • Yeasts or derivatives thereof A23L33/145 • • • Extracts A23L33/17 • • Amino acids, peptides or proteins A23L33/18 • • • Peptides; Protein hydrolysates A23L33/185 • • • Vegetable proteins A23L33/19 • • • Dairy proteins A23L33/195 • • • Proteins from microorganisms A23L33/21 • • Addition of substantially indigestible substances, e.g., dietary fibers A23L33/22 • • • Comminuted fibrous parts of plants, e.g., bagasse or pulp A23L33/24 • • • Cellulose or derivatives thereof
C07H SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS …	C07H3/00 Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms … C07H3/06 • Oligosaccharides …
C07K PEPTIDES (peptides in foodstuffs A23 …	C07K14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof C07K14/435 • from animals; from humans C07K14/46 • • from vertebrates C07K14/47 • • • from mammals C07K14/4701 • • • • {not used} C07K14/4732 • • • • • {Casein (in foodstuffs A23J)}
C08 ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON	C08B POLYSACCHARIDES; DERIVATIVES THEREOF
C11 ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES	C11C FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
C12N MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA …	C12N15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g., plasmids, or their isolation, preparation or purification; Use of hosts therefor … C12N15/09 • Recombinant DNA-technology

Business and markets for fermentation-derived food ingredients

Traditional, biomass fermentation, and precision fermentation may all be employed for producing food products and ingredients. There has been significant investment in fermentation companies, with total capital of $1.69 billion invested in 2021 (Good Food Institute 2021). The merits of choosing one type of fermentation technology over another has to be weighed against the availability of technology, costs, challenges in production and separation, regulatory hurdles, and likely consumer acceptance of the technologies.

The costs for precision fermentation have been falling exponentially since the first molecules from precision fermentation were produced. For example, proteins by precision fermentation is expected to be $10/kg by 2023–2025 and at this competitive price point, the market for food products may be unlocked (Tubb and Seba 2019). The cost of production for biomass fermentation also needs to be reduced for improved translation of technology for microalgae ingredients (Barros de Medeiros et al. 2022). A way to reduce the cost of biomass production is to reduce cultivation and harvesting costs. The principles used to reduce the cost of nutrients for cultivation, include the possible use of inexpensive resources as alternative sources of nutrients (e.g., waste or by-product streams as cheap and renewable carbon and nitrogen sources) (Ochsenreither et al. 2016), decreasing labor costs by increasing automation, and reducing power consumption for the production step (Acién et al. 2012). A recent review suggests that the development of high performance algal strains (e.g., through genetic engineering), and combining algae culture and low-cost harvesting with wastewater utilization, has potential for improved algal production systems (Xu et al. 2020).

Markets

Developments involving synthetic biology will be the next industrial revolution, attracting investment from private investments, mergers and acquisitions, minority stakes, and initial public offerings (BCG 2021). The convergence of increased accessibility to synthetic biology tools (e.g., DNA-sequencing, gene-editing), the developments in fermentation bioprocessing, the increased speed and testing solutions, and the exponentially reducing prices of these technologies (BCG 2021) have the potential to facilitate the development of transformative sustainable value chains based on precision fermentation.

In terms of fermentation-derived ingredients for human and animal nutrition, the strategic imperatives for success in the fermentation ingredient market over the period 2017–2022 were driven by the market, changing food habits, and the increasing demand for high-quality food products (Frost and Sullivan 2018). Products produced using synthetic biology tools have mainly been used in the healthcare, research, and industrial chemicals sectors, with the smaller segments being in food and beverage, agriculture, and consumer products (BCC 2022). In 2020, the global markets for synthetic biology in healthcare, research, and industrial chemicals were estimated to be $2.7 billion, $2.0 billion, and $1.5 billion respectively. The global markets for food and beverage, agriculture, and consumer products were $0.43 billion, $0.39 billion, and $0.35 billion respectively. The market for food and beverage is forecast to grow at compound annual growth rate (CAGR) of 51.3% and is estimated to be worth $5.7 billion by 2026 (BCC 2022).

The prospects for the growing markets for more sustainable and animal-free alternative food ingredients (Good Food Institute 2020) are driving interest in using precision fermentation technology. Genetically modified organisms have been used for improved yields and efficiencies of ingredients for the alternative meat and dairy sectors (Good Food Institute 2021; Hettinga and Bijl 2022). There has also been increasing attention paid to the engineering of microbial strains and fermentation strategies for producing colorants and flavors as alternative ingredients for the food industry (Seo and Jin 2022).

Fermentation companies

Fermentation companies have produced recombinant food additives/processing aids for commercial use in the food industry for some time. These include enzymes (e.g., chymosin for use in cheesemaking) (Olempska-Beer et al. 2006), and vitamins for food fortification (e.g., vitamin E) (Voigt 2020). Now, some of the largest food and life science companies, such as DSM, DuPont, Novozymes, and JBS are investing in fermentation-based product lines for the developing alternative protein industry (Pozas and Bushnell 2020). However, the interest in employing precision fermentation commercially for the emerging recombinant food ingredients has generally been the domain of new startup companies. Most precision fermentation activities of startups in recent years have been directed at the development of alternative proteins, although there are emerging companies with interests in recombinant lipids and oligosaccharides. This is due to the large emerging markets in the alternative animal-free protein for meat, dairy, and egg analogues. These markets are currently primarily served by plant-based analogues. However, there is potential for precision fermentation-derived proteins and lipids to capture more of the alternative meat and alternative dairy markets. Table 2 lists selected companies that are using fermentation for food-related businesses. These companies are at various stages of commercialization. The list spans companies with interests in developing strains for precision fermentation platforms, alternative animal-free proteins (e.g., egg-free and dairy-free) based on precision and biomass fermentation, lipids (e.g., omega-3 fatty acids from algae and fermentation-derived fats to replace animal fats and palm oil), and a range of other functional ingredients (e.g., prebiotics, post-biotics, and vitamins). Among the recent commercial food ingredients made by precision fermentation are (1) steviol glycosides for use as sweeteners (Hoeven Van Der, Galaev, and Boer 2015; Watson 2018; (2) soy leghemoglobulin produced by engineered yeast (Pichia pastoris) for use as a colorant and meat flavor/aroma enhancer in plant-based burgers and meat analogue products (Shankar and Hoyt 2016) (3) animal-free egg replacers (Southey 2021), and (4) animal-free dairy proteins (whey proteins and caseins) for the production of alternative dairy and formulated food products (Geistlinger et al. 2018).

Table 2. Selected list of companies involved in fermentation for production of food ingredients.*

Company	Interest – Ingredient /Product
	Strains/Recombinant Protein
Arzeda	Designer fermentation strains for ingredients
Peprotech	Recombinant proteins including growth factors for cultivated meat
Provenance Bio	Synbio tools for use in creating animal protein
Richcore Lifesciences	Recombinant proteins including growth factors for cultivated meat
	Proteins
Air Protein	Alternative meat and seafood using fermentation to transform CO2
AllG	Milk proteins through precision fermentation
Better Dairy	Production of dairy proteins using precision fermentation
Biosynthia	Mycelium as platform for animal muscle proteins in meat alternatives
Change Foods	Producing dairy proteins by fermentation for cheese
Clara Foods	Egg proteins through fermentation
Eden Brew	Milk proteins through fermentation
Final Foods	Whey proteins produced by yeast for cheese
Fumi Ingredients	Egg replacement ingredient produced by microbial fermentation
Geltor	Collagen/gelatin using fermentation platform
Harmony	Human milk proteins using fermentation for infant formula
Legendairy	Milk proteins through microbial fermentation for dairy products
Mayamilk	Milk proteins via precision fermentation
Motif FoodWorks	Algae-based burger patty; heme and other meat-like compounds for plant-based meat applications
New Culture	Caseins by fermentation for cheese production
Novacca	Milk proteins via fermentation platform
Perfect Day	Dairy protein using fermentation platform
Proprotein	Caseins from microorganisms
Remilk	Dairy proteins through microbial fermentation
String Bio	Converts methane for alternative protein applications
	Lipids
BioTork	Microalgae omega-3 fatty acids
C 16 Biosciences	Palm oil alternative produced by fermentation
Change Foods	Producing fats by fermentation for dairy products
Melt and Marble	Re-creating animal fats using precision fermentation
Nourish	Producing fermentation-derived fats for meat, dairy and fish alternatives
RhYme Biotechnology	Lipid-based ingredients by fermentation for alternative proteins
Yali-bio**	Precision fermentation-derived fats for animal-free products
	Other ingredients
Cargill-DSM	Steviol glycosides produced by fermentation
Cargill	Fermentation-derived postbiotics for food applications
DuPont	Probiotics for food, beverage and dietary supplements
DuPont	Vitamins for plant-based foods
Fybraworks Foods	Leg hemoglobulin for color and flavor in plant-based meats
Impossible Foods Phytolon	Functional ingredients for plant-based meats by fermentation Fermentation-based natural colors

* Sources: Company websites; Pivot Food Investment (2022).

** Poinski (2022).

Environmental, social and governance (ESG)

There is increasing scrutiny on ESG credentials for companies (Pavláková Dočekalová and Kocmanová 2018) as these non-financial indicators affect their economic value. Recent studies have shown that there is improved access to financial resources for companies with increased levels of ESG disclosures (Raimo et al. 2020), because of the relationship between ESG and financial performance (Buallay 2022). ESG information is particularly important for startups to access investment funds in the emergent precision fermentation industry and for these companies to grow and become a viable new business. However, there is still a need for development of standardized methods for assessing and reporting of benchmarked sustainability indicators and their wider acceptance for measuring corporate social responsibility.

Innovation pathway

After a market-led opportunity has been identified for a target food ingredient, a transdisciplinary team needs to be formed. Following the choice of the fermentation route for the target ingredient and definition of the functional requirements of the ingredient in food applications, an assessment of the science and patent information related to strain engineering for expression of the target ingredient and its recovery downstream from fermentation is reviewed. Together the information gathered is used to design the minimal viable product that can be launched in the market. This includes testing the ingredient in a target food application. The launch of the ingredient in the market-place requires prior investigation of food labeling and food regulations in the region where the product is intended to be marketed.

Innovation in precision fermentation-derived ingredients will be facilitated by leveraging new bio-manufacturing platforms (Zhang, Sun, and Ma 2017) and harnessing the benefits of new value chains created by opportunities in precision fermentation. Important examples of precision fermentation bio-manufacturing platforms include using engineered yeasts such as Pichia pastoris (Komogaetella phaffii) (e.g., for the production of animal-free egg proteins by The Every Company (Ivey et al. 2021a) and recombinant soy leghemoglobin by Impossible Foods (Shankar and Hoyt 2016), as well as engineered filamentous fungi (e.g., Trichoderma reesei) for production of recombinant milk proteins for animal-free dairy by Perfect Day (Pandya et al. 2016)). Engineered gram negative bacteria have also been applied as bio-manufacturing platforms for the recombinant production of collagen (Ouzounov 2017; Ouzounov et al. 2021), chymosin (Emtage et al. 1983), alpha-amylase (Zhang and Huo 2017), and human milk oligosaccharides (Jennewein and Wartenberg 2017). Considerable research efforts have focused on developing enhanced new bio-manufacturing platforms using genomic modifications to increase recombinant protein production and/or secretion (Bill 2014; Ivey et al. 2021a), to increase oil production (Dolch et al. 2017), to modify glycosylation (Ivey et al. 2021b), and to reduce allergenicity (Bhatt et al. 2021). Other examples of the new platforms include metabolic engineering and synthetic biology-driven whole cell fermentation (Zhang et al. 2017), conversion of waste plant oils or animal fats into value-added products by oleaginous yeast (Liu et al. 2021), building mevalonate and lycopene synthesis pathways in Escherichia coli to produce lycopene from glucose, fatty acid and waste cooking oil (Liu et al. 2020), and a combination of intracellular molecular engineering of Yarrowia lipolytica and extracellular fermentation, and bioreactor engineering for converting plant oils into high value products such as carotene, lycopene, wax esters, and omega-3 fatty acids (Xie et al. 2017). Irrespective of which type of fermentation platform is used, expertise in the chemistry and technology of the target ingredient will be required for developing appropriate isolation/separation processes. The characterization of the isolated ingredient and its application in food formulation will require food science and product development skills to get the ingredient to market.

Policy and regulatory status

The technology for production of new and novel fermented foods and food ingredients is emerging and continues to rapidly evolve. The commercialization of new and novel food products that have not had a history of safe use needs to be regulated in a transparent manner to ensure the safety of these foods when consumed. The risks and benefits of new products should be carefully considered. New regulations on different fermented food products are needed, with increased scientific evidence for safety, quality, and transparency (Mukherjee et al. 2022).

The diverse range of food products being explored and developed may use new sources of naturally occurring microorganisms/algae and genetically modified organisms, alternative sources of raw materials for fermentation (e.g., food waste), different fermentation process conditions, and downstream processing of fermentation-derived products. The EU General Food Law states that “Food shall not be placed on the market if it is unsafe” (van der Meulen 2012). Guidelines relating to the safety of microbial food cultures (i.e., live bacteria, yeasts or molds used in food products) are governed by frameworks which require “history of safe use”, “traditional food” or “generally regarded as safe”. The regulations on the safety of food cultures differ among different countries, although all are intended at assuring the safety of cultures that also need to be guaranteed by the food culture supplier (Laulund et al. 2017). An inventory of microorganisms used in food fermentation for various food matrices has been published (Bourdichon et al. 2012). The qualified presumption of safety (QPS), which is also “applicable to genetically modified organisms used for production purposes if the recipient strain qualifies for QPR status”, was developed to provide pre-assessment of safety risks and support safety risk assessments presented to ESFA (European Food Safety Authority). The list of QPS-recommended biological agents for food and feed use are updated regularly (Koutsoumanis et al. 2020).

There needs to be appropriate regulations, assessment of ecological and safety risks, and consideration of consumer concern relating to the use of genetically modified organisms for producing food and beverages. Scientists have a role to play in GMO regulation and they have to address and articulate uncertainty to policy makers (Myhr and Traavik 2002). Engaging the public in dialogue and addressing their concerns about GMOs (Doxzen and Henderson 2020) is necessary. The negative consumer attitudes toward foods produced using GMO for solving global food and nutrition security may be moderated through education, exposure to official information, and developing social trust (Du, Xiao, and Xu 2022; Mosier, Rimal, and Ruxton 2020). Table 3 summarizes the issues relating to the introduction of GMO foods, and actions that may be taken to address the public concerns of using GMOs and the policies to overcome concerns.

Table 3. Issues relating to use of GMOs in food and actions to address concerns.

Public concerns	Suggested actions	References
Gap between scientific organizations and public about safety of GMOs and their beliefs about the health and environmental impacts of GMOs	Involve general public to a greater extent in research carried out by universities and research organizations to increase confidence in science	Pappalardo, D’Amico, and Lusk (2021)
Ethical, cultural and religious concerns of GM technology	Address the importance of GM crops and improve public awareness	Abushal et al. (2021)
Societal concerns about safety (health and environment) CRISPR-edited foods	Improve science communication and increase public engagement in productive dialogue	Doxzen and Henderson (2020)
Inadequate representation of consumer preferences and policy incongruence	Include the voice of the customer when making laws to better represent consumer concerns	Mosier, Rimal, and Ruxton (2020)
Negative information concerning effects of GMOs and heat and environmental risks	Address knowledge gap of public; increase interactions between regulators and scientific community; communicate scientific research findings clearly and concisely to public	Paz and Zhang (2018)
Public concerns about the safety and efficacy of GM crops	Invest in better science communication and regulation to address unethical research and misinformation	Raman (2017)
Perception that GMOs have negative impacts on the environment and consumers	Continue collaboration between Government regulators and scientific experts – analyze how GMO products are introduced, weigh GMOs risks against judiciously	Yang and Chen (2016)
Public concerns about use, benefits, safety, potential effects on health and the environment	Improve public understanding of possibilities and limitations of GMOs through implementation of evidence-based educational programs	Rzymski and Królczyk (2016)
Regulatory challenge of considering interaction between scientific knowledge, risk assessment and management, policy, and public perception	Policy making needs to rely on the best scientific knowledge and meet transparency requirements; pro-active and transparent risk communication is important to address public concerns and educate the public about science advancements	Bartsch (2014)
Public concerns about level of risk, potential hazards, the lack of “naturalness” and what is communicated in the media by pressure groups	Governmental action to improve public trust in the regulatory process; independent processes to evaluate scientific evidence	Burke (2012)

The ability to navigate the regulatory framework has a significant influence on the transformative potential of novel ingredients, genetically modified or genetically edited ingredients and foods. Food laws and regulations to ensure food safety are complex for fermentation-derived food ingredients. Additionally, food regulatory frameworks are influenced by regional, political and cultural priorities, and these must be considered through integrated scientific and engagement with a range of stakeholders (Mukherjee et al. 2022), including the public. Understanding them, providing the level of evidence required to legislators, and including the cost and time of getting regulatory approval is a substantial undertaking (Lähteenmäki-Uutela et al. 2021). Apart from cost and time, there are additional issues including change in social, economic and legal systems, and the pre-cautionary approach adopted by government systems (Williams 2021). Mukherjee et al. (2022) suggest that each fermented product has to be regulated and include compositional specifications, safety, communication and distribution. Williams (2021) suggests that innovative regulatory governance needs to be more flexible and competitive to meet the regulatory demands and help promote the development and implementation of new foods. Strategies and actions directed at obtaining more harmonized and globally accepted regulations for fermented food products and ingredients will ease the path to market for new fermented foods and products.

Patent search results and analysis

The search identified 413 potentially relevant patent families. It should be noted that a patent family may have more than one aspect disclosed and therefore these appear in the listed categories several times and that patent information is not published in databases when a patent is first filed (i.e., at the priority date). The first publication of information for a patent may not occur for at least 18 months in most cases. Categorization based on the specific food ingredients of interest indicated the following number of patent families: proteins (231), lipids (81), carbohydrates (174), and all of proteins, lipids and carbohydrates (22). The top 10 assignees are DSM, Chr Hansen, PureCircle, BASF, DuPont Nutritional Biosciences, China Agricultural University, Danisco, Glycom, Ajimoto, and Evonik Degussa.

The main technology domains in the patent classifications are in “Biotechnology” (31.28%) and “Food chemistry” (30.32%). Other technology domains comprise “Organic fine chemistry” (10.53%), “Pharmaceuticals” (10.11%), “Basic materials chemistry” (5.00%), “Macromolecular chemistry/Polymers” (2.66%),”Other special machines” (2.45%), “Chemical engineering” (2.23%) and “Other miscellaneous domains” (5.42%). The concept clusters relating to the occurrence of terms in the patent families illustrates the distribution of the main concepts (Figure 2). Selected patents relevant to the development of fermentation-derived proteins, fat and carbohydrate ingredients are listed in Tables 4–6.

Figure 2. Concept cluster analysis of the occurrence of terms in patent families.

Table 4. Selected patents relevant to proteins from fermentation.

Inventions covered by selected patents	References
Recombinant Egg Protein
Producing an egg white protein composition, the method comprising recombinantly expressing two or more egg white proteins	Anchel (2016)
Ingredient composition for producing an egg-less food item, comprises a recombinant ovalbumin with egg white functionality	Mahadevan et al. (2021)
Recombinant Dairy Protein
Methods for manufacturing dairy substitutes (including methods of producing recombinant milk proteins)	Pandya et al. (2016)
Food product (e.g., cheese, yogurt) comprising one or more native and/or recombinant milk proteins and one or more native and/or recombinant non-animal proteins	Geistlinger et al. (2018)
Cheese and yogurt compositions and the methods of making the same using one or more recombinant proteins comprises a casein protein (but without beta-casein) and kappa-casein in the micellar form	Gibson, Radman, and Arie (2020)
Recombinant milk proteins with non-native post-translational modifications and food products containing recombinant milk proteins	Geistlinger et al. (2020)
A recombinant milk protein component consisting of a subset of whey milk proteins or of a subset of casein milk proteins for egg replacer	Geistlinger et al. (2022)
A micelle composition comprising an alpha casein and a kappa casein where at least one of the proteins is a recombinant protein	Radman et al. (2022)
A method for producing a casein micelle in a liquid medium containing calcium and phosphate, where at least one of the casein proteins is selected from the group consisting of recombinant αs1-casein, αs2-casein, β-casein and κ-casein	Wolff, Gaydar, and Cohavi (2022)
Recombinant Meat/Muscle Protein
Modified gram-negative bacteria comprising exogenous genes encoding target protein production (e.g., collagen)	Ouzounov (2017)
Methods and systems which combine synthetic biology, fermentation, material science and machine learning to produce collagen with desired physical and chemical properties	Persikov et al. (2019)
Synthetic meat made through recombinant expression of muscle proteins in edible biological hosts and crosslinking produced protein	Lu, Xia, and Liu (2021)
Recombinant Functional Ingredients
A cell comprising an exogenous nucleic acid molecule comprising a promoter sequence linked to a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding a heme - containing polypeptide	Fraser, Simon, and Brown (2017)
DNA constructs and methods of using such DNA constructs to genetically engineer cells for the production of heme-containing proteins	Roy-Chaudhuri and Shankar (2020)
Compositions with enzymatically active enzymes produced recombinantly, wherein the enzyme is a goose-type lysozyme for preservation of foodstuffs	Ivey, Kreps, and Harshal (2021)
A method for preparing a bovine myoglobin by constructing plasmids (containing genes for heme biosynthesis pathway enzymes and a gene for Bos taurus myoglobin) and constructing Escherichia coli production hosts and culturing. Product is useful as a meat flavor and/or an iron supplement.	Yoon et al. (2021a)
A method for preparing soy leghemoglobin by constructing plasmids (containing genes for heme biosynthesis enzyme pathways and gene for soy leg hemoglobin) and constructing E. coli production hosts and culturing. Product is useful as a meat flavor and/or an iron supplement.	Yoon et al. (2021b)
Biomass Fermentation / In-vitro Cell Cultures – Various Proteins
Producing single-cell protein from biomass by using at least one thermophilic fungal strain grown in a fermentable carbon-rich feedstock at a high temperature and at an acidic pH and recovery of the single-cell protein	De Latt and Gallego Murillo (2018)
Process for producing a cell hydrolysate composition, comprising substantially protein polypeptides and/or polypeptide fragments from in vitro cell cultures	Chin et al. (2021)
Systems and methods for producing cell cultured food products (sushi-grade fish meat, fish surimi, foie gras, and other food types)	Elfenbein and Kolbeck (2018)
A method of producing an isolated cultured milk product from mammary cells comprising culturing a cell construct in a bioreactor	Strickland (2021)
Processing microorganism cells from a culture medium to produce a food product (meat analogue food product)	Dyson, Rao, and Reed (2021)
General Methods – Recombinant proteins & Food Products
Systems and methods for the high-yield production of recombinant proteins in engineered microorganisms for expressing a heterologous protein	Ivey et al. (2021b)
A method for modification of protein glycosylation by recombinantly expressing a nutritional protein and an −1,2-mannosidase in a host cell, wherein the −1,2-mannosidase reduces the glycosylation of greater than 50% of the nutritional protein secreted from the host cell	Ivey et al. (2021b)
Recombinant milk protein having an attenuated or essentially eliminated allergenicity, where the recombinant milk protein retains one or more of the functional attributes of the native protein	Bhatt et al. (2021)
Food product produced by a recombinant host cell (wherein an activity of an esterase is essentially eliminated or modulated) and compositions comprising recombinant components for use in food products	Geistlinger et al. (2021)
Non-naturally occurring polypeptides comprises a sequence of a fragment of collagen and recombinant cells containing nucleic acid sequences for encoding them; Composition is formulated for consumption by human	Ouzounov, Mellin, and Co (2021)

Table 5. Selected patents relevant to lipids from fermentation.

Inventions covered by selected patents	References
Recombinant Lipids
A method for the production of docosahexaenoic acid in a microbial cell culture, wherein microbial cells have been transformed with a vector which encodes a polypeptide of a polyketide synthesizing system	Facciotti, Metz, and Lassner (2000)
Methods for the production of n-3 and/or n-6 fatty acids in a transformed oleaginous yeast containing the genes encoding a functional n-3/n −6 fatty acid biosynthetic pathway	Picataggio, Yadav, and Zhu (2004)
Construction and engineering of cells/ microorganisms for producing PUFAs with four or more double bonds from non-fatty acid substrates through heterologous expression of an oxygen requiring pathway	Forster, Gunnarsson, and Nielson (2005)
Engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing an oil containing >50 weight % eicosapentaenoic acid	Hong et al. (2010)
Polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems isolated from or derived from non-bacterial organisms, including nucleic acid molecules encoding biologically active domains of PUFA PKS systems for the production of bioactive molecules of interest (PUFA)	Metz et al. (2011)
Novel gene sequences and vectors useful for targeted modifications to the nuclear genome of heterotrophic microalgae in the manufacture of triglyceride oils	Franklin et al. (2012)
A method of manufacturing a triglyceride oil isolated from a cultivated oleaginous microorganism with an exogenous fatty acyl-ACP thioesterase gene that encodes a fatty acyl-ACP thioesterase having hydrolysis activity toward one or more fatty acyl-ACP substrates of chain length 8 to 18 carbon atoms, and produces triglyceride oil enriched in the chain lengths for which the thioesterase is specific	Franklin et al. (2013)
Producing triacylglycerols where some of the triacylglycerols have a short-chain fatty acid esterified at the sn-3 position using microbial cells which comprise exogenous polynucleotides encoding a fatty acid acyltransferase	El Tachy et al. (2021)
Biomass Fermentation
Process for producing a single-cell edible oil containing DHA by cultivating a marine microorganism (e.g., Crypthecodinium cohnii) in fermentors and recovering the oil with solvents	Kyle, Reeb, and Sicotte (1991)
A process for producing eukaryotic microbial lipids (particularly containing polyenoic fatty acids) comprises growing eukaryotic microorganisms in a fermentation medium to increase the biomass density	Reucker et al. (2001)
Algal flour comprising more than about 20% by dry weight triglyceride oil	Piechocki et al. (2011)
Microorganisms and methods for co-culturing photosynthetic microorganism with a heterotrophic microorganism to produce lipids	McNamara et al. (2018)

Table 6. Selected patents relevant to carbohydrates from fermentation.

Inventions covered by selected patents	References
Recombinant Oligosaccharides
Process for producing an oligosaccharide using a host microorganism and use of glycosidases in the purification of a produced desired oligosaccharide from a mixture	Jennewein (2015b)
A construction method for genetically engineering E.coli to obtain a high-yield strain for producing cold-adapted alpha-amylase and fermenting to produce alpha-amylase*	Zhang and Huo (2017)
Production of oligosaccharides in genetically modified bacterial host cells, and genetically modified host cells which comprises at least one recombinant glycosyltransferase, and at least one nucleic acid sequence coding for a protein enabling export of oligosaccharide	Jennewein and Wartenberg (2017)
Producing and purifying human milk oligosaccharides comprises fermentation of a genetically modified microbial organism, and downstream processing (with one or more treatments - enzymatic treatment/ filtration/ simulated moving bed chromatography)	Johnson and Janse (2019)
Genetically modified yeast cells capable of producing one or more human milk oligosaccharides (HMOs) and methods of making such cells to have heterologous nucleic acid encoding a transporter protein and heterologous nucleic acids that encode enzymes of an HMO biosynthetic pathway.	Walter and Pinel (2021)
Genetically modified microorganisms for enhanced utilization of oligosaccharides and improved productivity of compounds (tagatose, 2′-fucosyllactose, and psicose)	Chomvong, Cate, and Jin (2019)
Recovery and Separation for Recombinant Oligosaccharides
Process for the purification of neutral human milk oligosaccharides produced by microbial fermentation uses a combination of a cationic and anionic ion exchanger treatments, and a nanofiltration and/or electrodialysis step	Jennewein, (2015a)
A method for separating sialylated oligosaccharides from a fermentation broth produced by a genetically modified organisms. The steps include ultrafiltration, nanofiltration, activated charcoal treatment (optionally) and iv) treatment with strong anion and/or cation exchange resin	Matwiejuk et al. (2017)
Process for isolation of L-fucose from a fermentation broth. produced by microbial fermentation comprises removing biomass from the fermentation broth, subjecting the resulting solution to at least one of a cationic ion exchanger treatment and an anionic ion exchanger treatment and removing salts.	Helfrich and Jennewein (2019)
Large-scale purification of sialylated human milk oligosaccharides from microbial fermentation, using recombinant bacterial cells or yeast cells	Jennewein, Helfrich, and Engels (2019)
Preparing a purified human milk oligosaccharide (HMO) from an HMO solution by a process comprising nanofiltration, as well as processes for making foods, dietary supplements, medicines and infant formulas comprising a purified HMO.	Koivikko and Jarviene (2020)
Recombinant Functional Ingredients & Food Products
Polypeptides having an amylase activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides*	Callen et al. (2003)
Instant cereal composition prepared with addition of a liquid, containing at least one human milk oligosaccharide (HMO) for the growth and development of a young individual	Jennewein (2021)

* Included due to relevance to carbohydrate breakdown.

Precision fermentation technology – production of food ingredients

In precision fermentation, synthetic biology methods are used to program microbes which are used as cell factories to produce ingredients for the food and pharmaceutical industries (Pham 2018). The choice of the recombinant host microorganism and strain engineering present the initial challenge that determines the possibilities for constructing microbes to express and produce the target molecules in sufficient quantity using appropriate fermentation conditions for increasing efficiency of production. Microbial hosts are preferred for ease of genetic manipulation and use of standardized fermentation equipment. Microorganisms which are generally regarded as safe (GRAS) or have non-harmful status are preferred for food applications, and so strain engineering often utilizes benign bacteria (such as Bacillus spp.), yeasts (Vieira Gomes et al. 2018; such as Saccharomyces cerevisiae, Pichia pastoris (now Komagaetella phaffii), Kluyveromyces spp.), or filamentous fungi (such as Trichoderma spp., notably the popular T. reesei strains) (Chai et al., 2022; Nevalainen, Peterson, and Curach 2018). Innovation arises through the use of novel species or strains, or through utilizing genetic engineering and synthetic biology techniques to optimize the yield of the desired product, for example by improvements to expression, secretion, substrate conversion, and titer of the product. There is tremendous untapped potential within natural microbial biodiversity to provide efficient microbial cell factories for novel and safe fermented foods, which recent advances in “omics” tools and synthetic biology are uncovering. By utilizing these tools one can develop products that exactly fit desired properties and ensure that the fermentation process is precise and controlled rather than random (Teng et al. 2021). Precision fermentation lends itself readily to the production of target proteins, lipids, and carbohydrates, due to the ability to produce molecules that mimic the composition of their counterparts derived from traditional agriculture.

Production of precision fermentation-derived proteins

Whilst bacterial expression host E. coli has dominated the production of recombinant protein therapeutics due to well-characterized genetics, rapid growth and high yield (Huang, Lin, and Yang 2012), fermentation for protein food ingredients favors GRAS microorganisms which enable simpler regulatory pathways. Recent trends favoring the production of animal proteins using animal-free systems have led to a focus on eukaryotic expression hosts that are able to produce nature equivalent animal proteins with suitable post-translational modifications. Unicellular Crabtree-negative yeast such as Komagaetella phaffii (Pichia pastoris) provide an ideal expression system and have been widely used (Juturu and Wu 2018; Spohner et al. 2015; Vieira Gomes et al. 2018). A variety of genetic edits have been utilized to enhance recombinant protein production in this expression host including avoidance of protein degradation due to the unfolded protein response (Bankefa et al. 2018), enhanced processing of proteins through the endoplasmic reticulum for improved secretion yields (Zahrl, Mattanovich, and Gasser 2018) and deletion of unwanted protease activities (Vuree 2020). The development of genetic engineering tools such as CRISPR (Nishi et al. 2022; Prielhofer et al. 2017; Weninger et al. 2018) has enabled these synthetic biology approaches for recombinant protein production (Spohner et al. 2015; Wagner and Alper 2016). An early objective in the development of a recombinant protein is to obtain a nature-identical primary structure which has an amino acid sequence of the target protein to that found in nature. Post-translational modifications (e.g., phosphorylation, glycosylation) also need to be considered as they influence both the structure and functional attributes of a protein.

Innovative synthetic biology approaches to recombinant protein production for food ingredients are generally aimed at: (1) increasing protein expression per biomass unit (i.e., increasing titer and yield), (2) increasing the efficiency of protein trafficking through the cell, hence enhancing protein secretion, (3) enhancing specific physicochemical attributes of the recombinant protein product, such as thermostability (Cramer et al. 2018; Gautieri et al. 2018; Pietzsch et al. 2009) (4) protein engineering of specific functional attributes of the protein to create a desired food ingredient (e.g., altered catalytic substrate preference) (Prakash et al. 2020) or non-catalytic removal of allergens (Bhatt et al., 2021). Genetic modifications to achieve these aims are based on alteration of one to many genes through chromosomal substitution from a different donor organism, rendering a gene nonfunctional through partial or complete deletion, induced mutations to existing DNA or heterologous gene additions via episomal plasmid expression or chromosomal integration by recombination. For example, protein expression can be increased by manipulation of promotors and regulatory elements (Cai et al. 2019) or by deletion of extraneous proteins in order to direct cellular energy and protein expression systems toward the target product (Geistlinger et al. 2021). Protein titer in fermentation broth can also be increased by deletion of the more abundant proteases present in the fermentation broth. Increased efficiency of protein trafficking through the endoplasmic reticulum and Golgi body enhances protein secretion, for example through manipulation of the protein translocon and trafficking glycosylation signals (Ivey et al. 2021b). Additionally, redirecting recombinant proteins to avoid cellular degradation processes may be helpful in increasing protein secretion, for example by deletion of the vacuolar protein degradation transport system components in Saccharomyces cerevisiae (Bartkeviciuite and Sasnauskas 2003; Davydenko et al. 2004).

Selected patents on proteins derived from fermentation show that much of the recent patent activity is in the area of alternative proteins (Table 4). There has been significant interest in the development of recombinant egg, dairy, and meat proteins, and some activity in specialized functional ingredients (enzymes, color/flavor supplement) (Table 4). Animal-free egg replacers have been produced by precision fermentation (Anchel 2016; Mahadevan et al. 2021). Methods for producing recombinant caseins and whey proteins have been described, with some of the proteins produced having non-native post-translation modifications (Pandya et al. 2016). The recombinant milk proteins may be combined with other non-recombinant ingredients for the production of a range of dairy products such as milk, ice-cream, yogurt, and cheese (Geistlinger et al. 2018; Gibson, Radman, and Arie 2020). Recombinant milk proteins also have applications as an egg replacer (Geistlinger et al. 2022). With the use of synthetic biology tools, there are also possibilities to alter post-translational modifications (i.e., degree of glycosylation) of proteins (Ivey et al. 2021b), eliminate esterase activity which can be problematic for target food applications (Geistlinger et al., 2021), decrease allergenicity of protein (Bhatt et al. 2021) or produce non-naturally occurring polypeptides which comprise a fragment of a food protein (Ouzounov, Mellin, and Co 2021).

Other precision fermentation-derived ingredients for use as alternative proteins in non-animal meat analogues include recombinant collagen and muscle protein (Lu, Xia, and Liu 2021; Ouzounov, Mellin, and Co 2021; Persikov, Ouzounov, and Lorestani 2019). In addition, recombinant non-animal heme proteins are used to provide the color and flavor to meat analogues on cooking (Table 4). Alternatively fermentation-derived proteins and products have been produced by biomass fermentation or in-vitro cultures (Chin, Chan, and Poon 2021; Dyson, Rao, and Reed 2021; Elfenbein and Kolbeck 2018; Table 4). For example, milk has been produced by a mammary cell culture (Strickland 2021) and fungal single-cell protein has been used as an alternative protein in non-animal meat analogues (De Latt and Gallego Murillo 2018).

Production of fermentation-derived lipids

Oleaginous microorganisms such as yeasts, fungi, microalgae and thraustochytrids (e.g., Schizochytrium sp) accumulate lipids and produce polyunsaturated fatty acids which are beneficial for human health (Fan and Chen 2007; Patel et al., 2020). Improved yields of lipids in oleaginous microorganisms can be achieved by optimization of cultivation conditions and/or by use of genetically modified organisms (Béligon et al. 2016; Ma et al. 2021; Mahajan, Sengupta, and Sen 2019).

Table 5 provides selected examples of biomass fermentation for lipid production and gene editing methods employed for the production of a range of recombinant lipids. A wide range of synthetic biology approaches have been applied to enhance fatty acid and lipid production in fermentation, including enhancing the supply of substrate for acyl-CoA dependent systems, decoupling fatty acid production from growth, increasing carbon flux into the pentose phosphate pathway, and manipulating the expression of native and recombinant lipid-handling acyl transferases and thiol esterase enzymes to suit the desired lipid product (Table 5).

Most patent activity has been related to the production of polyunsaturated fatty acids (Forster, Gunnarsson, and Nielson 2005; Picataggio, Yadav, and Zhu 2004). Patented processes include those for the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from marine algae (Crypthecodinium cohnii) (Kyle et al. 1991), thraustochytrids (Facciotti, Metz, and Lassner 2000; Hong et al. 2010), and yeast (Hong et al. 2010; Picataggio et al. 2004). The health benefits of the long chain polyunsaturated fatty acids (EPA and DHA) (Shahidi and Ambigaipalan 2018; Swanson, Block, and Mousa 2012) have driven interests in these healthy fatty acids. Precision engineering has also been used for the production of triacylglycerols with short-chain fatty acids at the sn-1 and sn-3 positions (El Tachy et al. 2021). This provides a means to develop non-animal lipids that have a similar fatty acid profile as butter fat. A method for producing an alternative to palm oil has been developed by co-culturing a photosynthetic microorganism (e.g., cyanobacterium) with a heterotrophic microorganism (e.g., yeast) for the production of a lipid product with >40% palmitic acid (McNamara et al. 2018).

Pigmented oils have also been produced using yeast and microalgae. For example, the yeast, Phaffia rhodozyma, has the ability to synthesize astaxanthin as its major pigment, and may be used for commercial production as a biological source of carotenoids (Johnson 2003). The algal strains, Heamatococcus pluvialis and Chlorella zofingiensis, are considered ideal strains for commercial production of astaxanthin (Patel et al. 2022). Algal sources (e.g., red alage, green algae, brown algae, golden algae, yellow-green algae, dinoflagellates, and diatoms) are also producers of carotenoids (Patel et al. 2022; Schubert et al., 2006). The microalgae, Chlamydomonas reinhardtii, which has GRAS status may be used for producing carotenoid pigments and fatty acids. Recent studies have shown that the use of a gene edited strain, with optimization of the culture medium and process, enables co-production of pigments and oil (Song et al. 2022).

Production of fermentation-derived carbohydrates

In the field of carbohydrates, there has been most interest in the use of precision fermentation for the production of oligosaccharides. There has been a number of patents that describe the construction of genetically modified microbial cells for enhancing the production of oligosaccharides (Table 6). Human milk and related oligosaccharides, for application in infant formula and supplements, have been a primary target of interest. These oligosaccharides provide biological properties beyond nutrition to the infant including promotion of intestinal health, and the growth of beneficial microflora in the intestine (Ambrogi et al. 2021; Barile and Rastall 2013).

Precision fermentation derived food Ingredients – Downstream processing and scale-up

Downstream processing strategies vary depending on the type of ingredient and its ease of separation from the fermentation medium. Typical downstream processes for recovering of the target ingredient from the fermentation medium could involve cell lysis (depending on whether the ingredient is within the cell or secreted into the fermentation medium), filtration/centrifugation to separate cells/cell residues from the fermentation medium and separation processes typically used for food processing to isolate proteins, lipids or carbohydrate components from a complex mixture. There are two major pathways for downstream processing of fermentation products: recovery of intracellular products and recovery of products that are secreted into the fermentation broth. The first step for both pathways involve separation of cells from the fermentation broth. With the intracellular product recovery pathway there is an additional cell disruption step to obtain intracellular products, using methods combining one or more of heat, pressure, bead milling, enzymatic hydrolysis or chemical hydrolysis.

For the recovery of proteins after separation from the cell biomass, the major pathways follow the same essential processes: clarification (usually by centrifugation or flow filtration), extraction and purification, and usually drying into a powder (e.g., freeze-drying or spray-drying) (Fellows 2017; Warner 2019). Numerous methods are utilized for the extraction and purification of food proteins, following similar methodologies for protein recovery for other applications, with the exception that all processes must comply with relevant food grade standards. Most techniques for downstream processing of proteins are based on utilizing the differentiated biochemical characteristics of the product (such as pI, affinity, hydrophobicity, precipitation with specific reagents or at a specific pH) (Becker, Ogez, and Builder 1983). An initial diafiltration or tangential flow filtration step to concentrate the product into a suitable milieu is commonly followed by chromatography to extract or polish products, with the required degree of purity determining the number and type of chromatographic steps. For highly purified products, affinity chromatography followed by size exclusion chromatography are commonly used for protein purification, but for many food products only a simple one step chromatography extraction step is required (Behm et al., 2022; Labrou 2014; Raymond, Rolland, Gauthier, and Jolivet 1998). A consideration is the content of the protein and purity in the extract isolated from the cells required for functional performance. It is important to appreciate that each protein is unique and the medium into which the protein is expressed during fermentation and processing steps to recover the protein will all have an influence on the conformation of the protein and its equilibrium state, and consequences for functional behavior of the protein when used in food applications. More recently advanced computational techniques such as machine learning and artificial intelligence have been applied to the optimization of fermentation and downstream processing, enabling the parallel optimization of a plurality of different parameters (Pottinger et al. 2022).

The production of fermentation-derived lipids using non-genetically modified organisms have been in commercial production for some time. Processes for increasing biomass density for the production of microbial lipids in fermenters have been patented some time ago (Reucker et al. 2001). Production strategies and downstream processing for extraction and purification of single-cells oils are established (Ochsenreither et al., 2016; Ratledge et al. 2010). The oleaginous cells have to disrupted prior to extraction and downstream processing for recovery of lipids. This requires the use of efficient methods of cell disruption which enables cost-effective recovery of the lipid components (Senanayake and Fitchall, 2006). There are a number of methods to choose for cell disruption including (1) mechanical methods (e.g., bead milling, homogenization, ultrasound) and (2) non-mechanical disruption methods (e.g., decompression with pressurized gases, osmotic shock, microwaves, drying, and chemical methods such as application of solvents, and use of enzymes). The method of cell disruption will influence the complexity of the extraction process for recovery of the lipid. After cell disruption, the lipids for food industry applications are extracted with nontoxic solvents (e.g., supercritical CO₂) or without solvents (Ochsenreither et al. 2016).

As for recovery and separation of recombinant oligosaccharides, the processes for purification include the removal of biomass from the fermentation broth, and a combination of filtration steps (e.g., ultrafiltration, nanofiltration) and ion exchange treatments (Helfrich and Jennewein 2019; Jennewein 2015a; Matwiejuk et al., 2017).

Application of precision Fermentation-Derived ingredients in food

Both the science and the patent literature help to provide the basis for ingredient development that leads to potential exploitation of market-led opportunities of fermentation-derived ingredients. After the expression of the target food ingredient/component, methods for separating the ingredient from the fermentation broth are carried out to recover the target ingredient. Unlike ingredients for pharmaceutical use where a high degree of purity if required, other components from the medium that are co-extracted with the target ingredient may have a positive or negative effect on functionality and this needs to be assessed in the final food application.

The major food ingredients (proteins, lipids, and carbohydrates) each have their own unique physico-chemical characteristics, nutritional properties, and techno-functionality, which are sought after for various food applications. Understanding the properties and functionality of traditional ingredients is required to inform fermentation targets and potential applications in food. The composition of the ingredient is a primary determinant of its functionality, although it may be modified by processing.

Briefly, the physical functional properties of proteins that are useful in food applications include solubility, heat stability, gelling, viscosity building, foaming, and emulsifications (Foegeding and Davis 2011). Some of the physical functional properties of fat that are important for food applications include their crystallization and melting behavior (Sato and Ueno 2014) and quality of oil for frying applications (Choe and Min 2007). Polyunsaturated fatty acids, such as omega-3 fatty acids, are nutritionally desirable (Shahidi and Ambigaipalan 2018). Due to their susceptibility to oxidation which leads to the production of off-odors and off-flavors, unsaturated fatty acids need to be protected during isolation and processing. The carbohydrate biopolymers are sought after for their water-holding and texture-modifying properties (Yang et al. 2020). Prebiotic carbohydrates/prebiotic fibers (e.g., oligosaccharides) are of interest because of their nutritional role and health-promoting properties (Elleuch et al. 2011) and ability to contribute to the physical properties of foods.

Further information on properties and functionality of food ingredients, which is beyond the scope of this review, is available. Some relevant reviews of the characteristics of these major food ingredients and their functional properties include those for proteins (Augustin and Udabage 2007; Day 2013; Kristo and Corredig 2015; Małecki, Muszyński, and Sołowiej 2021; Phillips, Whitehead, and Kinsella 1994), lipid analysis (Christie and Han 2012) and food lipids (Akoh 2017; Eskin and List 2017; Gunstone and Norris 1983), carbohydrates (Gao et al. 2017; Li and Nie 2016; Nishinari and Doi 1993) and prebiotics (Sungsoo and Finocchiaro 2009).

Key issues for commercialization of precision fermentation technology

It is acknowledged that there are fermentation-derived ingredients already in the market. However, there are a number of issues that need to be considered and addressed for realizing the full potential of fermentation-derived ingredients. High product yields and use of cost-effective culture media are important for the economic viability of the process, as are policy, regulations and consumer acceptance. There are recent patents that describe systems and methods for the high-yield production of recombinant protein (Ivey et al. 2021a). From a technology point of view, alternative fermentation feedstock and scaling of the fermentation process are barriers that need to be overcome for improved implementation of technology for fermentation. These issues are discussed below.

Fermentation feedstock

Cost-effective and sustainable fermentation feedstocks as alternatives to sugar are being sought for fermentation. Corn steep liquor, raw sugar extracts, and crude glycerin are inexpensive by-products that can be used to replace costly traditional media ingredients for precision fermentation of lactobacilli and yeasts (Gullón et al. 2008; Warner 2019). The use of alternatives to glucose as the carbon source for the microbial production of lipid has been explored (Sijtsma, Anderson, and Ratledge et al. 2010).

There is potential for exploitation of new substrates such as by-products and waste from the food industry. Nutrients have been recovered from food waste and used as alternatives to the use of traditional culture media (Pleissner et al. 2013). While lignocellulosic materials are abundant and generally low-cost, pre-processing of agricultural and food waste may be necessary to release the fermentable sugars (Nieves, Panyon, and Wang 2015). Some examples include the use of pretreated rapeseed meal for microbial oil production by Rhodosporidium toruloides (Uçkun Kiran et al. 2012) and pretreated brewers’ spent grains as a fermentation media for Rhodosporidium toruloides, a natural yeast producing carotenoids (Cooray, Lee, and Chen 2017). By-products of processing have also been explored as possible low-cost raw materials for generic microbial feedstock after a bioconversion process involving enzyme and/or fungal pretreatments. Agricultural by-products such as rice bran, wheat straw, sugarcane bagasse, fruit waste, tomato pomace, grape must, and corn cobs can all be utilized as low-cost culture media components for microbial precision fermentation production systems, albeit sometimes requiring pretreatment with cellulolytic fungi such as Trichoderma spp. (Raghuwanshi et al. 2014; Wang, Zhai, and Geng 2020). For yeast bio-manufacturing platforms that cannot naturally utilize sucrose, such as Pichia pastoris, engineered expression of an invertase enzyme(s) to convert sucrose to dextrose, with addition of suitable sucrose transporters for cellular uptake for enhanced efficiency are required for the utilization of raw sugar (Martínez et al. 2017). Food waste, such as wasted bread many be used as a growth medium for the cultivation of food industry microbial starters (Verni et al. 2020).

The logistics and costs for transporting agricultural and food waste to fermentation facilities for use as feedstock will need to be considered. This makes proximity of fermentation facilities to agricultural residues or food manufacturing plants with usable by-products of processing an attractive proposition. The effects of substitution of alternative feedstocks for sugar on the microbial metabolic pathways and food safety of using these alternatives will need to be evaluated.

Scaling of the fermentation process

For precision fermentation to reach its full potential, the ability to scale fermentation processes and isolate the target molecule is critical. The scale-up for precision fermentation is acknowledged as being a major hurdle that needs to be addressed in translation of fermentation technology to commercial scale. There is limited information on how the microorganisms adapt to living in a bioreactor and the interplay between the ecology and evolution of the process (de Lorenzo and Couto 2019). There are uncertainties in predicting the behavior of microbes when moving from a laboratory scale to larger scale, which require an improved understanding of the interactions between the physiology of the cell and the environment in larger-scale bioreactors (Delvigne et al. 2017).

A recent review discusses the design of efficient microbial cell factories that couple systems metabolic engineering and bioprocesses for industry. There are challenges to the industrial scale production and recovery processes for fermentation-derived compounds, which require integration of upstream (strain development), midstream (fermentation) and downstream (recovery and purification) aspects through the lens of system efficiency (Rangel et al. 2020). Irrespective of whether microbes are used for the conversion of substrates into energy, biofuels or production of compounds, there is a need to improve microbes for sustainable biotechnological processes, and this requires robust and very productive microorganisms. The capacity to use inexpensive and readily available fermentation feedstock is also needed for the economic production of fermentation-derived compounds. There is a need for the development of improved microorganisms which can drive the carbon flux toward the desired pathway, have increased tolerance to toxic compounds, and wider substrate uptake range, as well as the ability to generate new products (Paes and Almeida 2014). There should be an integrated metabolic-separation and purification view, which examines the growth medium for the microorganism, the effects of metabolic engineering on by-product generation, and the consequences on the cost of separation and purification processes (Rangel et al., 2020).

While research and development for a number of precision fermentation-derived proteins, fats and carbohydrates may have been proven on a laboratory and pilot scale, there are still bottlenecks for their production on a commercial scale. There is insufficient commercial fermentation capacity on a global scale to meet the forecasted fermentation demand for the production of precision derived ingredients. For commercial processing, there are options to build new purpose-built facilities for fermentation and downstream processing (Greenfield construction) which are fit for purpose but have a longer timeline and higher cost compared to a Brownfield retrofit. The latter involves retrofit of existing fermentation facilities and building new purpose build downstream processing facilities, which could cost less but still requires substantial capital (Warner 2021).

Conclusion

Fermentation for the production of food ingredients to feed a growing population in a more sustainable manner requires a whole new way of thinking about future food value chains. Precision fermentation is an example of a disruptive technology platform that offers the promise of more sustainable food production. There are technical costs, regulatory, and commercialization considerations, as well as the understanding and willingness of the consumer to use nontraditional ingredients. There is also a need to develop and use agreed methodology for substantiating claims relating to the sustainability of the technology. The full potential of using fermentation for the production of the majority of food ingredients has yet to be explored and realized. Adoption of fermentation technology can be facilitated by bringing together of expertise in synthetic biology, traditional, biomass and precision fermentation, scale-up from laboratory to larger scale, downstream processing for recovery of products, and food science to enable the delivery of consumer acceptable food ingredients. Through-chain integration of sustainable ingredient supplies which are acceptable to the consumer, and supported by regulation and policies, are sought after by the market. This will require both the involvement of multiple stakeholders and a transdisciplinary approach to innovation (Knorr and Augustin 2021; McClements et al. 2021) in precision fermentation-derived food ingredients.

Acknowledgements

The authors would like to acknowledge Dr Martin Cole for helpful comments and suggestions.

Disclosure statement

The authors do not have any competing interests.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.