Fermenting Forward: Tradition meets Trend

Fermentation has a rich and diverse history in the context of food production, and involves the production of fermented foods and food ingredients, the latter through both traditional and more modern, precision fermentation processes. Significant scientific and technological advances have taken place over centuries, which have transformed the production of fermented foods from art to science, and this is likely to continue as their dietary and nutritional role is increased, and as the world becomes more multicultural. On the other hand, precision fermentation offers a considerable opportunity to innovate in order to address important societal and environmental food system challenges1, 2

Fermented foods have been part of human diet for more than 7,000 years and were among the first ‘processed foods’ produced as the means for extending the shelf life of raw materials and drastically modifying their organoleptic and textural properties. The first fermented foods included beverages; wine and beer were made in pre-historic China and Sumeria as early as 4000 BC, respectively, whereas soy sauce was first made in China in the form of a thick paste called jiang and was originally a blend of soybean, meat, grains and salt used as a condiment to preserve food and to enhance its flavour as at that time salt was an expensive commodity¹. Bread production is evidenced in 4000 BC by the Egyptians who discovered that carbon dioxide generated through the fermentation process could leaven bread, whereas ‘Dahi’, a yoghurt-like fermented milk product of India, was mentioned in about 6000 to 4000 BC in ancient sacred books of the Hindus². The humans of the pre-historic period made the discovery that spontaneously fermented foods could be stored for longer than the raw materials and sometimes they tasted better. Over the years, they developed knowledge on how to control the environmental conditions (e.g. storage, process) and the type and properties of the raw materials to produce consistent fermented products of good quality, and this resulted in the development of small scale production; an example of this is a 4,500-year-old bakery discovered near the pyramids of Giza. Fermentation became a widespread production practice during the Roman Empire and by the Middle Ages, the production of fermented foods had become an established trade activity, often carried by monks in monasteries. The key scientific discoveries that provided the basis of fermentation industrialisation during the 18^th and 19^th centuries and the significant increase in the volume and complexity of operations and products, was the discovery by Louis Pasteur that each type of fermentation was mediated by a specific microorganism. To this end, he described in a series of scientific publications the lactic acid fermentation and ethanol fermentation pathways focusing on the metabolic activities of lactic acid bacteria and yeast, respectively. He also discovered that oxygen inhibits glycolytic metabolism, which paved the way for further studies on microbial physiology investigating the response of microbes to the environment where they grow in (e.g. pH, temperature, aeration, nutrients); these formed the knowledge-base for industrial fermentation.

By the middle of the 20^th, century a significant microbial starter culture industry was established, initially serving primarily the bakery, brewery and dairy sectors (and later on the meat, vegetable, soy sauce sectors), producing a range of starter cultures, including lactic acid bacteria (e.g. Lactobacillus delbrueckii, Lactococcus lactis, Pediococcus acidilactici, Leuconostoc mesenteroides), yeast (Saccharomyces cerevisiae) and moulds (e.g. Penicillium roqueforti, Aspergillus awamori).

The fermentation industry grew alongside the increase in popularity of fermented foods and the advances in process engineering, large scale biomanufacturing and plant design. For example, in the beginning of the 1900s, starter cultures were produced as liquid cultures but due to issues with over-acidification during production which led to loss of cell viability during storage, alternative technologies were developed and optimised for production and storage; these included the production of frozen bulk and concentrated cultures as well as freeze dried cultures, involving cryoprotectant agents such as glycerol, sucrose and lactose. Nowadays, the production of starter cultures involves the use of large scale bioreactors (>10,000 litres) and downstream processing (e.g. continuous centrifugation, freezing, drying) in factories operating under good manufacturing practices (GMP), with a significant degree of process automation and stringent quality control procedures. In parallel to the advances in manufacturing, the significant advances in genomics and molecular biology over the last 40 years have considerably accelerated product and bioprocess development. Following the complete genome sequence of Saccharomyces cerevisiae in 1996³ and Lactococcus lactis in 2001⁴, several commercially important strains have been sequenced since. The integration of this knowledge with next-generation sequencing, multiomics (transcriptomics, proteomics, metabolomics), bioinformatics tools, metabolic control analysis, and targeted and untargeted metabolic product analysis such as Nuclear Magnetic Resonance (NMR) and High-Resolution Mass Spectrometry (HRMS), provides unique capabilities to screen, select, and rationally design, strains with desired properties⁵. These include increased cell growth rates, reproducible performance, bacteriophage resistance (bacteriophage is a virus that infects bacteria and is most the most common type of contamination in commercial fermentation processes, particularly in dairy), production of desired flavours and aromas and textures, lack of off-flavours, tolerance to osmotic and temperature stresses, production of functional and bioactive components (e.g. vitamins, polysaccharides, antimicrobials, minerals, peptides, polyphenols).

The potential nutritional and health benefits of fermented foods (particularly lactic acid fermented foods), as well as the increasing uptake of plant-based diets, is an important reason for their cultural popularity, which has increased considerably over recent years. This surging demand is reflected in the sales of fermented foods and beverages, with the global market projected to reach $989.2 billion by 2032⁶.

The link between lactic acid fermented foods and human health was initially proposed by Nobel prize winner Ellie Metchnikoff in his book ‘The prolongation of life’⁷. He attributed the increased life expectancy of Balkan people to the significant consumption of yoghurt, and associated this to the high amounts of lactic acid produced by the Bulgarian Bacillus strain used for production (classified now as Lactobacillus delbrueckii subsp. bulgaricus). According to Metchnikoff, this strain ‘acclimatised in the digestive tube for the purpose of arresting putrefactions and pernicious fermentations, such as the butyric fermentation’. Following on from these initial observations and hypotheses, a substantial amount of follow-on work has shown that the ingestion of lactic acid fermented foods can modulate the gut microbiome in terms of its functionality and its response to stress and attacks, due to the presence of lactic acid bacteria species and their metabolites produced during fermentation, including antimicrobials, bacteriocins, organics acids, signalling molecules and B-vitamins⁸. However, the consensus is that a higher number of well conducted in vivo human trials are needed on a wider population to provide a better understanding of the potential beneficial impact of fermented foods, the likely mechanisms involved, and potentially formulate dietary recommendations. Such studies should include popular foods, like yoghurt, cheese and wine, but also indigenous fermented foods produced in various parts of the world, as well as industrialised products that are expanding at a global scale, such as kombucha, kefir and kimchi.

Non-dairy and meat-free fermented foods that are produced using plant-based ingredients (e.g. soy, vegetables) are perceived to be natural, clean-label and nutritious alternatives to animal-derived products. Potential benefits include increased digestibility, lower allergen content, increased bioavailability, enhanced nutritional value, and removal of antinutrients (e.g. enzyme inhibitors, oxalate, phytic acid, tannins). Popular vegan fermented products include water kefir, a sparkling acidic drink originating from Mexico, as well as cheese and yoghurt analogues made from plant-based proteins. Fermented foods widely consumed and marketed as meat analogues, include tempeh and tofu (both soybean products), seitan (produced using wheat gluten) and Quorn (mycoprotein). Further studies are needed to provide a better understanding of the role of the microbial strain, processing strategy (with the view to minimise processing) and ingredient composition on the sensory and nutritional properties of meat analogues, aiming to improve the qualities of these products⁹.

The continued expansion of our global population, the rise in climate-related disruptions and the decrease in available agricultural land, present a compounding set of challenges endangering current food supply. Innovations in food processing and production can enable our food system to meet our growing demands, while minimising adverse impacts on the environment, economy, and society. Microbial-based solutions have been garnering increasing interest in the context of food production, given their potential to produce plant- or animal-derived nutrients with a significantly reduced environmental footprint.

In recent years, the production of food ingredients using engineered microorganisms as ‘cell factories’ has been termed precision fermentation. What differs precision fermentation from traditional microbial biotechnology, historically used in food production for the production of ingredients (e.g. organic acids, amino acids), is the concept of metabolic engineering - the use of genetic engineering to manipulate the metabolism of a microorganism, such as bacteria, yeast or mould with generally regarded as safe (GRAS) status - to produce the products of interest. Notably, metabolic engineering unlocks the range of molecules of interest that can be industrially produced via fermentation. The process of developing an engineered strain generally consists of identifying the biosynthetic pathway of the desired product, such as a protein or vitamin, and re-constructing the same in a microbial host, by introducing genes found in the plant or animal origin species¹⁰. Commercialisation of a precision fermentation process requires it to reach the right yield, productivity, titre and economy factors that hinge on the native traits of the selected host and the optimisation of the desired metabolic pathway¹¹. Rapid development of genetic engineering and synthetic biology tools, have enabled the implementation of a multitude of strategies for yield improvement. The intersection of computational biology, protein engineering, multi-omics, automation and synthetic biology continues to give rise to more efficient strain development¹⁰. As in the case of natural strains, engineered strains once established, are cultivated in a bioreactor, generally requiring a carbon source such as glucose, nutrients and air to proliferate and produce the compound of interest. The product is then recovered, separated from the cell biomass, and purified, making it ready to be used as a food ingredient¹².

As of today, precision fermentation has been used to produce macronutrients (proteins, carbohydrates, lipids), vitamins, additives (e.g. flavouring agents, texturisers, sweeteners, colourants, preservatives), and a variety of other nutraceuticals¹¹. A pivotal example is the production of riboflavin or vitamin B2, where precision fermentation has replaced chemical synthesis since the 1990s¹³. Within the alternative protein and dairy space, major achievements include Impossible Foods’ soy leghaemoglobin production by engineered Komagatella phaffi (formerly Pichia pastoris), Perfect Day's β-lactoglobulin (milk protein) for ice cream products produced using engineered fungus Trichoderma reesei and the EVERY Company demonstrating egg-white protein synthesis from engineered K. phaffi¹².

A key driver for commercialisation, evidenced by these examples, is the ability of precision fermentation to ensure safe food production, at any location, which is sustainable. Products made by fermentation have reduced reliance on land, lower greenhouse emission and use less water compared to the traditional industrial methods for sourcing them such as agriculture, bulk extraction, husbandry and organic synthesis¹³. In the case of specific animal proteins, precision fermentation is forecasted to be as much as 100 times more land efficient¹². However, a key bottleneck affecting both the economic feasibility and sustainability of biomanufacturing, is feedstock use. One key area of focus for metabolic engineers is optimising the use of inexpensive sustainable feedstocks, such as side streams from various industries including food by-products, agricultural food-grade lignocellulosic residues and by-products including rice bran and wheat straw¹¹. Engineering of photosynthetic hosts such as cyanobacteria could further be used to sequester carbon dioxide whilst producing high-value ingredients¹⁴. This valorisation of residues and by-products, showcases the potential of precision fermentation to enable the realisation of a circular bioeconomy. Beyond environmental benefits, this technology can be an essential tool for maintaining stable and sufficient supply of vital nutritional elements, such as human milk oligosaccharides, unique prebiotic components of human breast milk¹⁵. As such, precision fermentation holds the potential of being one pillar upholding a more sustainable and resilient future food system.