The science of fermentation

https://gfi.org/science/the-science-of-fermentation/

Learn about the emerging role of microbial fermentation in building the next generation of alternative protein products.

Tiny organisms, big potential

Fermentation has been used in food production for millennia. Ancient civilizations used microbial cultures to preserve foods, create alcoholic beverages, and improve the nutritional value and bioavailability of foods ranging from kimchi to tempeh. Over the past century, the role of fermentation has expanded far beyond its historical usage to a much broader range of applications.

Fermentation now spans industrial chemistry, biomaterials, therapeutics and medicine, fuels, and advanced food ingredients. The suite of tools developed through fermentation’s evolution is now poised to revolutionize the food sector by accelerating the rise of alternative proteins.

The term “fermentation” carries distinct meanings across different disciplines. Within biology, it refers to a specific metabolic pathway used to generate energy in the absence of oxygen. Within the alternative protein industry, fermentation is used in three primary ways:
Traditional fermentation uses intact live microorganisms to modulate and process plant-derived ingredients.

Traditional fermentation results in products with unique flavor and nutritional profiles and modified texture. Examples are using the fungus Rhizopus to ferment soybeans into tempeh, as well as using various lactic acid bacteria to produce cheese and yogurt. There are also more modern renditions of this concept, such as MycoTechnology’s fermentation of plant-based proteins to improve flavor and functionality.
Biomass fermentation leverages the fast growth and high protein content of many microorganisms to efficiently produce large quantities of protein.

The microbial biomass itself can serve as an ingredient, with the cells intact or minimally processed — for example, the cells can be broken open to improve digestibility or enrich for even higher protein content.

This biomass serves as the main ingredient of a food product or as one of several primary ingredients in a blend. Examples of biomass fermentation are Quorn’s and Meati’s use of filamentous fungi as the base for their products.
Precision fermentation uses microbial hosts as “cell factories” for producing specific functional ingredients.

These ingredients typically require greater purity than the primary protein ingredients and are incorporated at much lower levels. These functional ingredients can improve sensory characteristics and functional attributes of plant-based products or cultivated meat.

Precision fermentation can produce enzymes, flavoring agents, vitamins, natural pigments, and fats. Examples include Perfect Day’s dairy proteins, Clara Foods’ egg proteins, and Impossible Foods’ heme protein.
Innovations are occurring across all three types of fermentation.

Despite microbial fermentation’s long history in food and industrial biotechnology, tremendous potential for innovation remains untapped. The vast biological diversity of microbial species, coupled with virtually limitless biological synthesis capabilities, translates to immense opportunity for novel alternative protein solutions to emerge from fermentation-based approaches.

Opportunities for advancing fermentation can be segmented into five key areas spanning the value chain: target selection and design, strain development, feedstock optimization, bioprocess design, and end-product formulation and manufacturing.
Two-tone icon of a double helix, representing dna
Target selection and design

When microorganisms are used as production hosts to create specific high-value ingredients, identifying and designing the right target molecules to manufacture is key.
The current state of target selection

Biology provides food developers with an almost boundless palette of molecules from which to assemble flavors, textures, and aromas. However, not all these ingredients are easily sourced at large volumes and low prices. By using microbial cells as the production host, precision fermentation allows for highly scalable manufacture of virtually any ingredient.

Target selection and design is the starting point for the process of precision fermentation. The molecule or molecules of interest are referred to as the target. The target can be a protein, a lipid, a flavor compound, a fragrance, an enzyme, a growth factor, a pigment, or another class of molecule.
Fermentation-derived ingredients are already widely used across the food industry.

The majority of vitamins in nutritional supplements and fortified processed foods, such as B12 and riboflavin, are produced through fermentation, as are many flavoring components. The food industry was among the first to leverage fermentation to displace animal products in everyday use.

Commercialization of fermentation-produced chymosin (the major enzyme in calf rennet, taken from the lining of calves’ stomachs) in the 1980s rendered calf rennet’s previously vital use as a coagulant in cheesemaking obsolete for most global cheese production.

A notable recent example of precision fermentation’s use in alternative proteins is Impossible Foods’ use of purified soy leghemoglobin. “Heme,” produced using Pichia pastoris, is used as a flavoring ingredient in their burger to produce a suite of organoleptic properties in the cooked product.

Other recombinant proteins, such as casein and whey, are key targets because of their unique functionality in dairy products. These proteins can be combined with plant-derived ingredients to create a final product. For example, sugar, coconut oil, and sunflower oil are combined with fermentation-produced recombinant whey to make Perfect Day’s ice cream base.
Precision fermentation targets specific molecules.

Target molecules such as animal-origin-free growth factors are used in the production of cultivated meat. Several companies, including ORF Genetics, Richcore, and Peprotech, already work in this space. Furthermore, proteins such as collagen or fibronectin produced through fermentation may serve as key animal-free components of scaffolding for more complex, highly-structured cultivated meat products.

In the case of a protein target, the instruction manual for synthesizing the protein is encoded in the host organism’s DNA, either as a naturally occurring gene or as a gene introduced through engineering. Depending on the target, both engineered and non-engineered approaches may be possible.

For example, the soy leghemoglobin protein produced by Impossible Foods is engineered into a yeast host strain for efficient, scalable production. On the other hand, microalgae company Triton Algae Innovations is commercializing heme proteins that are native to their algal strains, so no engineering is involved.

Synthesis of non-protein targets cannot be encoded directly in the host’s DNA. Instead, the genome encodes a series of enzymes that compose the biosynthetic pathway for producing the target molecules.

For example, the target molecules for algal omega-3 production are the fatty acids DHA and EPA, but the instruction manual for manufacturing these fatty acids consists of several gene-encoded enzymes that convert precursor fatty acids into these desirable fatty acids within the cell. As with protein targets, molecules like fats or flavoring molecules can be produced in microbial hosts either with or without the use of engineering techniques, depending on the specific target and the choice of host organism.
Challenges in target selection for precision fermentation

One of the most basic challenges for target selection is simply determining which molecules contribute the most to specific properties of animal products. A litany of volatile compounds, many of which differ by species type and cut, contribute to the taste of different kinds of meat. These compounds should be more holistically characterized and cataloged to develop a consensus “wish list” of target molecules as candidates for production through fermentation.
Mass-producing already-existing molecules

In many cases, several variants of a candidate target may already exist in nature. For example, almost every living organism contains heme proteins of some sort, but which ones perform the best as flavor enhancers for meat products? Which are the most stable — not only during their production, but also through the downstream processing of the final food product and throughout its shelf life? Which target accumulates at the highest titers within host cells, thus allowing for the most favorable economics?

All of these answers must be ascertained through a combination of thorough empirical screening and predictive approaches.

For target molecules that are not proteins, there are additional challenges: identifying biosynthetic pathways that can manufacture these molecules, and then determining whether these pathways already exist in suitable host organisms or if they must be engineered or enhanced for higher productivity.

For example, fermentation-derived lipid production is relatively unexplored for food applications but has a fairly robust history for industrial chemicals. The alternative protein industry may be able to develop an open-access research foundation and accelerate the commercialization of fermentation-derived fats by aggregating lipid synthesis pathway insights from the chemicals industry.

Each of these aspects feed into one of the key challenges within precision fermentation: improving the economics of production. To compete with animal-based proteins, researchers and companies must increase the titer (amount of an expressed target molecule relative to the volume of total upstream-produced liquid containing the agent – the primary benchmark of upstream efficiency) and yield (the ratio of the mass of final purified protein relative to its mass at the start of purification – the primary benchmark of downstream efficiency) of target molecules and protein biomass.

While strain development and feedstock optimization can contribute substantially to the overall economics, the target selection is a critical factor in achieving economic viability.
Where target selection innovation for precision fermentation is headed

Fermentation allows for a decoupling of the original source of a target molecule and its production method. This decoupling vastly expands the search landscape for biomolecules with unique and valuable functions.

First, ideal targets may originate in species that are extraordinarily rare, difficult to harvest, expensive, or otherwise inaccessible or impractical. Fermentation provides a mechanism for manufacturing these molecules at scales and prices suitable for commercial viability.

Second, targets are not limited to those found in nature: Novel variants of target molecules can be engineered through random alteration and screening (directed evolution) or through rational design, leading to targets that exceed the performance of any naturally occurring version.
Mastodon collagen anyone?

Geltor’s collagen production platform illustrates both aspects of this expanded search landscape. Gelatin (a form of collagen) from conventional animal sources is limited to a few species (predominantly pig and cow, although fish gelatin is also commercially available) that are processed in large quantities. But collagen is ubiquitous in the animal kingdom, and Geltor can manufacture collagen proteins from any species, including extinct species.

In 2018, the company showcased the versatility of their platform with an animal-free leather binding made from jellyfish collagen and

characteristics desired for a particular application — for example, gelatin that exhibits a specific gelling viscosity, elasticity, or melt temperature.

Similarly, fermentation allows for enzymes to be adapted or engineered to exhibit higher activity, novel substrate specificity, greater stability, or robustness under specific processing conditions, with dramatic implications for cost reduction. Such enzymes serve many purposes across the alternative protein industry.

These examples show that fermentation holds immense potential to screen for natural variants of targets and to design new variants for augmented sensory, functional, or nutritional properties or for attributes that reduce costs and streamline manufacturing processes.
Check out related resources
Explore research opportunities in target selection

Fermentation icon Fermentation Plant-based icon Plant-Based 

Affordable animal-free omega-3 ingredients for alternative seafood and other alternative protein applications

In order to appeal to health-conscious consumers, alternative seafood products should contain similar omega-3 fatty acids, especially DHA and EPA, content to conventional seafood. Animal-free omega-3 ingredients can be expensive…
Read more

Fermentation icon Fermentation 

Novel methods for long-chain omega-3 fatty acid production

As the alternative seafood industry scales up, a low-cost and abundant source of long-chain omega-3 polyunsaturated fatty acids will become necessary. Several means of producing these compounds have been investigated…
Read more

Cultivated icon Cultivated Fermentation icon Fermentation Plant-based icon Plant-Based 

Preventing oxidation of omega-3 fatty acids before and after addition to alternative seafood products