Summary & highlights Experts Meeting Fermentation Process Improvement & Development of Novel Microbial Ingredients

The Fermentation Process Improvement & Development of Novel Microbial Ingredients experts meeting was held on Thursday May 6, 2021. The meeting hosts were Dr Derek Butler and Dr Radhika Bongoni from BaseClear. The meeting focused on how genomic technologies are being used to improve fermentation processes and develop new ingredients in the food and pharmaceutical industries. Each session was followed by a lively interactive panel discussion.

Session 1: Development of novel microbial ingredients

Session 2: New technologies for process measurements

KEYNOTE: Omics-based approaches for antibiotic discovery in Streptomyces cell factories

Antibiotic resistance of pathogens threatens our ability to treat bacterial disease. Regular news stories cover the threat of running out of effective drugs against common bacterial pathogens (see story “WHO sounds alarm on drug-resistant bacteria,” April 2021. How can genomics help us to discover and develop new antibiotics in the war against antibiotic-resistant “superbugs”?

Currently, pharmaceutical companies have done a good job of finding effective antibiotics. The “low-hanging fruit” has been discovered. However, 95% of the biochemical space is as-yet unexplored. Genomics can help out both on the discovery and process development arenas of new product development.

As an example, the Streptomycetes are bacteria found often in soil or decaying plant matter that produce a large number of secondary metabolites that are potentially active against other bacteria. Streptomyces coelicolor produces actinorhodin, a blue pigmented antibiotic that has not been commercialized (see article by Mak and Nodwell). It can serve as a model organism for how genomic technologies can help identify antibiotics.

Sequencing the complete genome of Streptomyces coelicolor in 2001 was a major breakthrough in uncovering the secrets of the microorganism (see article from Dr David Hopwood on the history of Streptomyces coelicolor research). Indeed, the now rapid and affordable genomics technologies that have developed over the past 20 years have been enormously beneficial in many areas of academic and industrial research. Streptomyces coelicolor contains a wealth of different metabolites or enzymes to allow it to adapt to changes and compete in soil, however the majority of the genes that express these proteins are “sleeping” unless exposed to the right conditions to stimulate their expression. Metabolites produced in small quantities can also be difficult to produce for further study. By sequencing the genome of Streptomyces coelicolor, metabolites that are “sleeping” under laboratory cultivation can be discovered and the trigger elucidated.

Streptomyces in their natural environment are able to use different carbon sources for growth. The surrounding concentration of different carbon sources is used by the microbe to control certain growth characteristics: growth is promoted under carbon source abundance, while their absence triggers metabolite production. Carbon sources can be seen as signalling molecules. One such molecule, N-acetylglucosamine, has been investigated recently in the laboratory of Dr van Wezel, who used genomic information to guide the research. This molecule forms part of the cell wall of many organisms, including fungi, insects and spiders, which are common soil residents. Streptomycetes preferentially uses N-acetylglucosamine as a carbon and nitrogen source. It acts as a switch to encourage the production of metabolites including antibiotics. Presumably, a selection advantage could arise by preventing growth of other bacteria when this carbon source is present.

A co-culture plate that includes different pathogenic bacteria, Streptomyces coelicolor and the inducer N-acetylglucosamine can be used to screen for the production of useful metabolites. Furthermore, the in silico identification of genes involved in N-acetylglucosamine metabolism can assist in the production of mutants with differing effects on growth and antibiotic production. The discovery of elicitors can be used to re-screen different bacteria and fungi for production of useful metabolites that were not produced under the initial test conditions. Whole genomes can be screened to identify metabolite-producing organisms and their metabolic triggers.

The integration of genomic technologies including metabolomics, enzyme structural analysis, and in vitro testing was used in the characterization of a novel antibiotic that is produced in low concentrations by a Streptomyces strain (see report from Xiao). Insights generated from this work are particularly important for the production of angucycline analogues or the general laboratory growth characteristics.

Genomic technologies can also help to improve yield within the bioreactor. While Streptomyces produce a large number of potentially useful metabolites, they are not well suited for high-volume production of those metabolites. In particular, the vegetative hyphae show a clumping behaviour that increases viscosity within the fermenter, which reduces oxygen transfer and can be a nuisance for down-stream processing.

The protein SsgA is important in Streptomyces for the development of aerial hyphae and sporulation. SsgA overexpression produced a reduction in clumping behaviour, whilst SsgA-knockout mutants produced large clumps of mycelium in liquid culture (see van Wezel et al.). This could improve growth and yield in submerged cultivation. Likewise, reverse engineering allowed the identification of the matA and matB genes that also influenced clumping behaviour in Streptomyces lividans. Bioinformatics was used to perform homology searches to predict the function of the products of these genes. Ultimately, as reported in Microbial Cell by van Dissel and co-workers, an extracellular polysaccharide was found to be catalysed by matAB, changes in which could affect pellet formation.

Taken together, Streptomycetes show enormous potential for the discovery of new antibiotics. Omics-based techniques are important over the entire road to production: from discovery to fermentation at industrial scale.

Presentation 2: Strain adaptation for whey permeate fermentation

Whey, a waste from cheese production, can be converted to whey protein and lactose, however whey permeate leftover from these processes has limited market potential and is often disposed of as animal feed. Whey permeate disposal is a bottleneck for whey processing. It is a great challenge for the dairy industry in general to increase the value of this waste stream.

The ash content of whey permeate is the most used metric in terms of waste streams. The current focus is on three areas: lactose recovery using fractional purification, fermentation of lactose to lactic acid, and recovery of lactic acid from fermentation broth.

A process using chemical precipitation, membrane technology and lactose recovery removes half to three quarters of ash from the waste stream. The relatively high osmotic pressure of the whey permeate limits fermentation. A sequential fermentation system using de-lactosed whey permeate was used to select strains that were able to grow well under increasing osmotic pressure. The efficiency of conversion and growth rate were able to be maintained. Using the strain found using this method enabled a much higher lactic acid yield, increasing the value of whey permeate.

The process for the recovery and purification of lactic acid from whey permeate now generates several co-products in addition to lactic acid. The pre-treatment step yields calcium phosphate, which can be used for human and animal nutrition. The microbe used to convert lactose in the waste stream can be sold as a probiotic. Gypsum produced during the acidification step produces gypsum, used in mushroom compost and as a soil conditioner. The final product polylactic acid is a bioplastic. This process is part of AgriChemWay, a consortium developing valuable co-products in a sustainable way from whey permeate.

Presentation 3: Clean label optimisation of plant-based products by application of fermentation technology

Plant-based foods are currently receiving considerable interest from consumers, key opinion leaders for sustainability, nutrition, and industry. The protein transition has several drivers:

Switching to plant protein potentially results in lower overall land use for food production.

Science into plant-based foods focuses on several strategies to improve the use of plant-based foods. New sources of plant foods increases the “menu” of options available. Protein extraction efficiency could be improved within food processing to extract more edible protein. Plant-based food quality and taste is important for their acceptance by consumers. Novel proteins need to be rigorously tested for safety and approved for use by regulatory bodies before they enter the food supply.

For consumers directly, cost is important: plant foods should be at least similarly priced as the animal protein. Clean label issues can hamper the adoption of plant foods by astute consumers. Some plant foods can have unexpected flavours that should be addressed in the final product. New products may lack information on shelf-life or regulatory approval processes, or may have additional shelf-life issues that are currently unknown.
Fermentation allows the production of a whole range of different products that are useful in plant-based protein products. As well as being a source of protein themselves, microbes can sustainably increase the nutrient content of plant foods with vitamins and fatty acids, increase their shelf life through production of preservatives and antioxidants, or modify flavour or sweetness. Yeast extracts are very important for improving palatability. Flavour tuning such as enhancing the sweetness perception of the product, and adding “meaty” flavours or culinary notes such as vinegar or spicy sensations to food are possible with yeast extracts.

In terms of alternative biomass protein production, there are a number of directions being developed currently. Microbial platforms include bacteria, yeasts, fungi and animal stem cells. In many cases the cost of the final product for consumers is unclear due to the high cost of raw ingredients. For example, yeast extract is used in many fermentation applications but is a relatively expensive ingredient. An additional limitation is that the full Life Cycle Analysis of some production processes indicate that they also have a considerable environmental impact that is similar to animal-based protein.

An example of how microbes can be used to change the sensory properties of food is from soy beans. Although they can be consumed raw or as tofu, two traditional fermentation-based soy products, natto and tempeh, have a completely different texture and taste profile. Using different types of microbes for the fermentation produces these disparate results. In a more modern example, acidification and heat treatment of casein allows the addition of soy to milk without unduly affecting the texture.

Many different high throughput analysis tools can help with selecting strains and fermentation conditions that enhance properties of plant foods. For example, consumers do not necessary expect or appreciate the soy flavour in soy yoghurt. Off-flavours in soy arise from aldehydes present. Fermentation can reduce the aldehydes to alcohols, thereby reducing the “beany” taste in soy yoghurt.

Food spoilage is also a problem that can be addressed with fermentation. This is traditionally seen in dairy and meats, that use lactic acid bacteria as a starter culture to increase shelf life in products such as cheese and salami. Modelling technologies can help determine optimal preservation conditions.

Presentation 4: Crowd control of a zillion cells

Bioprocess optimisation is an important part of R&D on dairy cultures and probiotics. Classical strain improvement, modern biotechnologies and screening are an important part of strain discovery. Production requires strain conservation, fermentation, formulation and downstream processing steps. Sensory and probiotic properties are important for the consumer. Analytical capabilities and sensory attribute analysis enables the development of a new product.

Bioprocess optimisation is a major part of dairy culture and probiotic production to make a high quality product with a high yield. Differences between laboratory, pilot and production scale can be a challenge during new product development: differences in medium sterilisation, fermenter design and downstream processing equipment can influence the final product. It can help to keep conditions as similar as possible between different scales to reduce unexpected effects. However, some effects of scale-up cannot be held the same due to the larger size.

Starter cultures have different purposes. Many manufacturers in large companies use an undefined mixed bulk starter culture. This can be a challenge as every fermentation is a little bit different. Some Lactobacillus bacteria are used as a flavour adjunct. Streptococcus thermophilus is used for yoghurt and mozzarella.

Lactose in milk is converted to lactic acid in different dairy foods to prevent spoilage, enhance taste and modify the texture through the formation of polysaccharides. Important parameters are temperature, pH and the addition of a base. These environmental factors much be controlled in a similar way to ensure reproducibility between batches or within a continuous fermentation system. The equipment used is a major factor in how well the process is controlled.

The size of the tank has a large effect on effective mixing time. The stirrer type and position of base dosage are also critical factors. A grid model can help calculate what happens inside the fermenter and also to identify the best location for sensors that steer the fermentation. Quality control parameters such as optical density, cell count and acidification speed determine fermentation performance.

The sedimentation properties of dairy cultures are important for downstream processing. Stoke’s Law uses bacterial size, particle density, the density of the medium, the viscosity of the medium and gravitational force to estimate sedimentation rate. The measurement of fermentation broth optical density during centrifugation provides a good estimate of the sedimentation properties of fermentation broth.

A limitation in cheese research is the long ripening time needed to assess flavour development. For many medium and hard cheese, a cheese trial requires 6 months of ripening, which slows the process of innovation. Sensory testing machines can assist by tracking changes in ripening parameters over time to give an earlier estimate of flavour development.

Presentation 5: Genomics technologies for fermentation applications

How can new technologies be used in different fermentation applications? Genomics includes culturomics and strain discovery, and strain characterisation of viruses, bacteria and fungi. Strain characterisation includes microbial identification and typing, draft genome and resequencing, strain collection resequencing, closed genome, safety assessment and regulatory affairs issues. In addition, it may include gene expression profiling (including transcriptomics), bacterial community profiling and shotgun metagenomics. There are many examples of applications using genomics technologies within fermentation.

As shown from these case studies, there are multiple ways that genomic analyses can support every aspect of fermentation. These technologies can provide support particularly in the critical points in bringing a product to market: regulatory approval, production strain stability, and working cell bank purity.

Presentation 6: Genomics in beverages: The good, the bad and the brew

The clear trend in beverage production globally are in low- and no-alcohol beers and in alcoholic beverages, associated to health and wellness. Another is the rise in home-brewing, craft beers and variation in flavours and bitterness in beers. The low alcohol content in beers and ciders makes them more prone to microbial contamination. Knowledge of fermentation and brewing is needed to fully meet market expectations of fermented beverages.

Non-conventional brewing microbes contain several interesting qualities that support these trends. They are able to modify the fermentation process to provide a new sensory experience by varying the flavour, texture and acidity of beer. They can also produce antimicrobial substances naturally to improve shelf-life. Probiotic or prebiotic effects, or improving the nutrient content of beverages, may also be possible. Food-grade yeasts, acetic acid and lactic acid bacteria could potentially provide some of these benefits.

Genomics has several applications in the development of novel fermented beverages. It can be used to understand conventional brewer’s yeast, the different strains used and their genotypes. This can help in strain selection and development. Spoilage microorganisms and their detection within the production process or biofilms can be facilitated with genomics.

Artisanal starters are a clear part of new fermented beverage trends, providing interest and potential new flavours to beer. Metagenomics is a way to identify components and key microbes in artisanal starters. Genome sequencing and annotation can further refine the search to the species level, and discover useful genes. High throughput screening allows strains with particular benefits to be identified, also those lacking negative traits. Genomics is also essential for food safety verification. Some trends here are cocoa fermentation, tea fermentation, and the mixed fermentation used for kombucha.

Genomics was used for the genotyping of 157 commercial Saccharomyces cerevisiae brewers’ yeasts in the publication Gallone et al. The analysis showed that the brewing strains tended to cluster together, separate from the wild type strains. Phenotypes important for brewing including stress tolerance, sugar use and flavour production show the effect of industry selection, whilst the sexual cycle is decayed. Another publication from Peter and co-workers surveyed a much larger group of 1,011 Saccharomyces cerevisiae isolates, which provides information on the history and domestication of the yeast. Changes in copy number produce variety of phenotype. This means that it is easy to generate variants within the strain. Natural strain crossing can also be a source of innovation, and genomics can document these changes.

Beer spoilage organisms are a challenge within the industry. Technologies such as genome sequencing, gene mapping, transcriptomics, metabolomics, mutant analysis and gene transfer can help with identifying spoilage organisms and using the knowledge to prevent their growth. In particular, beer spoilage organisms in the environment are often below the limit of detection with standard techniques. They can only be found via enrichment. A wide variety of microbes can cause spoilage, and genomics can help to devise hygiene strategies.

In identifying novel brewing strains, the genome of other food-grade microbes can be analysed. For example, Acetobacter pasteurianus 386B is an acetic acid bacterium identified from cocoa fermentation. A genomic analysis conducted by Illeghems, Vuyst and Weckxv has identified strain-specific genes for novel enzymes and tolerance to certain environmental conditions. The species is also a beer spoilage organism. The genomic analysis, and therefore knowledge of stress resistance and metabolites produced, could help in preventing contamination of beer batches.

When using novel strains, bacteria or yeasts in fermented beverage production, safety is very important. Also, proof of safety is required for regulatory approval. Genomics can show the absence of genes that can form a safety issue, such as toxin production or antibiotic resistance. The presence of other genes, and genetic stability can also be demonstrated with genomics. The decision tree in the article by Parziza and colleagues shows genomic analysis as an important initial step in proving the safety of a fermentation product.

In the future, genomic technologies such as next generation sequencing, metagenomics and transcriptomics will find more applications. This is particularly the case for quality, safety verification and product development.

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