Tackling resistance to food preservatives: Heterogeneity in fungal spore populations

Up to one third of all foods is spoiled by fungi. This is a major concern for global food security. Spoilage of food by fungi renders the food inedible, and potentially toxic due to formation of substances termed mycotoxins. Fungal spores are abundant in the environment and prolific contaminants, being able to develop and grow in many conditions. Despite a variety of strategies to preserve foods, spoilage microorganisms fight back and some develop resistance to particular preservatives. Soft drinks are typically acidic and these conditions tend to inhibit other organisms like bacteria. However, many species of fungi (especially the moulds) are able to grow in acidic conditions and cause spoilage. A main preservative strategy is to include weak acids like sorbic acid to inhibit growth under these conditions. At permitted levels, sorbic acid inhibits most yeasts and fungi, but a number can still grow and some can degrade the sorbic acid to non-toxic products which alter flavour. Consequently, a chronic level of mould spoilage occurs in the manufacture of soft drinks. Our work has shown that most spoilage fungi produce a small subpopulation of spores that are highly resistant to sorbic acid. Spores produced by other fungi seem to be more uniformly sensitive to the preservative. It is this ability to produce a subset of spores that are hyper-resistant, responsible for fungal growth at higher sorbic acid concentrations, which is the focus of this project. We will apply the latest genetic technologies to single spores to find out what defines these hyper-resistant spores. We will then exploit that information to develop novel methods for tracking these spores in laboratory media and beverages. Finally, we will apply these new tools to find alternative agents that can inhibit the sorbic acid-resistant spores. This approach could offer solutions for reformulating preservatives to give more complete inhibition of spoilage moulds, an area of particular interest to our industrial partners supporting this project. While soft drinks and sorbic acid resistance provide the exemplar for this work, the knowledge generated will help develop strategies for preventing fungal food spoilage more broadly.

The hypothesis to be tested in this study is that resistance of fungi to weak acid preservatives is traceable through individual, phenotypically-variant spores. The hypothesis has been formulated from our current and previous studies, funded by the BBSRC and industrial partners. We have shown striking population-heterogeneity in the outgrowth of asexual fungal spores when sorbic acid is present. This heterogeneity within the genetically-uniform populations is rooted in the spore stage. At sorbic acid concentrations permitted in foods, rare hyper-resistant spores may seed growth of spoilage fungi. The new objective is to characterise, then trace these spores so that we can find novel ways to inhibit them effectively. We will exploit our expertise in fungal-spore transcriptomics, this time applying innovative molecular-indexing technology to probe variation among individual spores. This approach combined with the timely availability of filamentous-fungal deletant libraries together with our existing knowledge of weak acid action, will be used to characterise variably-expressed genes responsible for heterogeneous preservative resistance. We will use these insights to develop fluorescent reporters for tracking resistant spore subpopulations. Finally, these tools will be put to use in finding and testing agents that may selectively target the sorbic acid resistant spores, so offering opportunities for reformulating preservatives. The research will concentrate primarily on the model spoilage mould Aspergillus niger, but extended also to other weak acid preservatives and other moulds isolated from spoiled beverages, of primary interest to our industrial partners.

Who will benefit from this research?

This proposal is for an Industrial Partnership Award, so we are already engaged with industrial partners who stand to benefit from this research. L.R.Suntory Ltd in particular is contributing funds to support their interest and involvement, as it wishes to improve anti-fungal preservation practices for its soft drinks products like Ribena and Lucozade. Mologic Ltd is providing in-kind support, as it has interests in fungal diversity in industrial plants and microbial diagnostics for health. The research could improve overall production efficiency and so economic competitiveness of a number of companies in the food and beverage industry. These companies currently have to deal with complaints and take remedial action. In addition to the focus of this proposal on food spoilage, the knowledge generated on heterogeneity in fungal-populations is also relevant to fungal disease and its control. For example, phenotypically-variant spores may seed infection or drug resistance. Therefore, bodies concerned with animal and human health including the pharmaceutical industry could benefit. As well as benefitting industry, the improved products from those industries and associated food security will of course benefit consumers, i.e., the general public.

How will they benefit from this research?

This research will contribute to improving the nation's health and wealth. Fungal spores are the starting point to contamination of crops, humans and other animals, foods and beverages. The outgrowth of fungal spores is the critical stage in the production of mycelia and infection or spoilage. However, the spores are not all identical. This heterogeneity makes problematic fungi more difficult to control, as we have shown with preservative-treated spores of spoilage fungi. This research focuses on that spore population-heterogeneity with a view to informing the development of more effective preservatives to prevent mycelial outgrowth. In its final stage, this project encompasses the design and testing of novel preservative formulations. Companies will benefit from decreased risk of spoilage and of off-flavours from products of fungal sorbic acid-degradation like 1,3-pentadiene, and so decreased risk of product loss. A product recall following customer complaints can cost a company several millions of pounds. The general public will benefit from the decreased risk of consuming food products that are contaminated and improved food security. Knowledge on the basis for spore-spore heterogeneity could also help in the fight against fungal pathogens, by directing drug development to target subpopulations that may be drug resistant. Finally, the researchers employed on this project will gain from interfacing between industry and academic research, with skills that will increase their employability and benefit UK industry.