Spoilage-yeast metabolism, reduced-sugar formulations and implications for food preservation

Fungi (yeasts and moulds) spoil 5-10% of all food and drinks produced globally, costing many billions of pounds each year. This contributes significantly to food insecurity. A variety of strategies is used to preserve foods but these are not always effective. In the soft drinks industry, formulations have been developed over a number of years that help to limit the spoilage problem to current levels. The drinks are usually acidic and usually inhibit bacteria. However, yeasts can grow in these conditions and they commonly dominate in soft drinks spoilage. A principal preservative used in soft drinks is the weak acid sorbic acid. This inhibits most yeasts, but a number can still grow at the permitted levels and some can degrade the sorbic acid to products that alter flavour. The resultant chronic level of yeast spoilage in soft drinks manufacture could be set to worsen, as recent market pressures have been prompting major re-formulation of soft drinks products. The 'Soft Drinks Industry Levy' implemented by the UK government in 2018 aligned with similar moves made by certain other governments. As a result, many manufacturers have decreased the sugar content of drinks formulations from more than 10% to less than 5%. However, there is little understanding of how these re-formulations may impact preservative efficacy and spoilage. Reports from the industry indicate a rise in incidence of certain spoilage yeasts since introduction of the reduced-sugar products. Our preliminary studies have illustrated how metabolism of spoilage yeasts is markedly altered by changes in sugar content below 5%. The yeasts shift from a type of metabolism termed fermentation to another, respiration, as sugar level is decreased. Importantly, this shift coincides with marked changes in the yeasts' abilities to resist preservative. Furthermore, these effects are not the same for all individual yeast cells in a population, a phenomenon known as 'heteroresistance', which can be a particular problem for spoilage control. Besides reduced sugar, there is also growing market pressure to use natural products in place of chemical preservatives, for cleaner label drinks products. This project focuses on understanding the impacts of these changes in drinks formulations for yeast metabolism and preservative resistance, and for spoilage control. We will investigate this by testing yeasts in low sugar conditions, which can be precisely controlled using a technology known as microfluidics. This technology also allows us to examine single yeast cells, as some individual cells can be highly resistant to preservative. We will apply the latest genetic technology to single cells to find out what makes them resistant and we will then exploit that information to find alternative agents that could give better inhibition at different sugar levels. Importantly, this will encompass tests of candidate natural-product activities, including some promising candidates from our industry partner supporting this project. The proposed project could offer solutions to give more-complete inhibition of spoilage yeasts in new formulations, an area of particular interest to the industry partner. While soft drinks and resistance to preservatives (including natural products) at different sugar levels provide the exemplar for this work, the knowledge generated will help develop strategies for preventing yeast food spoilage more broadly.

This study will test and exploit the hypothesis that metabolism and preservative (hetero-)resistance of spoilage yeasts is altered with reduced sugar. This tackles issues arising from soft drinks re-formulations recently undertaken by the industry, to align with the UK government's sugar levy since 2018. It addresses also wider food industry trends. The hypothesis stems from our recent work funded by the BBSRC and industrial partners. Our data suggest that, with decreasing sugar, a shift from fermentative to respiratory metabolism of spoilage yeasts coincides with sensitization to the preservative sorbic acid. However, this effect is not the same for all cells within a population, indicating heteroresistance. At sorbic acid concentrations permitted in foods, rare hyper-resistant cells may seed spoilage. We will capitalise on microfluidics technology to control the cells' environments and test whether a more potent, respiration-targeting preservative action emerges at low glucose. Besides sorbic acid, we will test a novel antifungal activity in juice concentrates from our industrial partner. Such candidate natural products are highly sought as potential alternatives to chemical preservatives. Our second objective focuses on the basis for preservative heteroresistance. We will use yeast genetic tools and recent molecular-indexing technology to probe single-cell variation at varying sugar. This will indicate differentially-expressed genes that drive heteroresistance, enabling us to make constructs for purifying the resistant cell subpopulations. We will exploit this tool for finding agents, including natural products, which may target sorbic acid resistant cells at different sugar levels. The results will show how recent pressures on drinks formulations are impacting preservative efficacy, and how we can exploit this knowledge for improved spoilage control. We will concentrate on model spoilage yeasts, but extend also to other yeasts isolated from spoiled beverages.

Who will benefit from this research?

This proposal is supported by industrial partner L.R.Suntory, a major international producer of soft drinks including Lucozade and Ribena. L.R.Suntory is investing in this research in order to improve anti-fungal preservation practices for its soft drink products. The focus of this application on yeast metabolism and preservative resistance at low glucose is particularly timely given the introduction of a Sugar Levy by the UK government in 2018, as manufacturers have been encouraged to reformulate their products. These companies currently have to deal with complaints and take remedial action in response to spoilage, which have high associated costs. New spoilage problems arising in post-levy drinks formulations have principally involved yeasts, the focus of this application to tackle the problem. 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. The research could improve overall production efficiency in the soft drinks industry and, as a result, improve economic competitiveness of a number of companies. It will also help drive the development of novel natural product preservatives, which meet the needs of consumers for clean-label products. The general public will benefit from the decreased risk of consuming food products that are contaminated and improved food security. The results may also encourage other manufacturers to reformulate products and hence the introduction of new low-sugar options in the marketplace. This will help in the effort to tackle health problems associated with high sugar diets. 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.