Tackling resistance to food preservatives: Heterogeneity in fungal spore populations

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Hewitt S (2016) Phenotypic heterogeneity in fungi: Importance and methodology in Fungal Biology Reviews

 
Description Evidence for variable chitin content of individual fungal spores, suggestion that spore size may affect weak acid resistance, protection against sorbic acid preservative by N-acetyl cysteine or ascorbic acid (vitamin C), dataset for A.fumigatus kinase mutants that are sorbic acid sensitive, accumulating evidence of whether MIC or heterogeneity in response to sorbic acid is related to food spoilage propensity of fungi. Evidence of mitochondrial and respiratory actions of sorbic acid against fungi. Deveolopment of single-spore transcriptomic method. Novel transcription factor that appears to regulate sorbic acid resistance of fungi, and heterogeneity in that resistance. Combinations with other agents that appear to work synergistically with sorbic acid in inhibiting fungi. Evidence for respiratory targeting by sorbic acid, so fermentative yeasts are more resistant. Respiration also seems to promote RNA mistranslation, a novel possibel mode of preservative action.
Exploitation Route Suppression of sorbic acid preservation by ascorbic acid (or other antioxidant) content of soft drinks could inform new formulations to help prevent spoilage and waste. Similarly, new insight to mode of action and synergy between sorbic acid and other agents could be exploited to improve food shelf lives.
Sectors Agriculture

Food and Drink

Healthcare

 
Description We are finding some novel combinations of agents which could have synergistic preservative action in foods against spoilage fungi. We have helped to inform changes in practice at LRS. Findings informed drink re-formulation, factory hygiene, and etc. as well as understanding of fungal population heterogeneity, with impacts on product spoilage and subsequently factory practices and subsequently product spoilage. Crucial for helping understand the mode of action of preservatives on fungi and explore alternative 'natural preservatives' in line with clean label trends.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Societal

Economic

 
Description BBSRC IAA
Amount £29,500 (GBP)
Funding ID BB/S506758/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 06/2022
 
Description Biodiversity and heterogeneity of spoilage yeasts in relation to food preservation hurdles
Amount £47,000 (GBP)
Organisation Unilever 
Sector Private
Country United Kingdom
Start 09/2019 
End 09/2023
 
Description Biodiversity and heterogeneity of spoilage yeasts in relation to food preservation hurdles
Amount £100,279 (GBP)
Funding ID BB/T508949/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2019 
End 09/2023
 
Description EPSRC-GCRF Pump Priming
Amount £11,940 (GBP)
Funding ID EP/R512849/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 03/2019
 
Description Improving the nutritional quality of future protein
Amount £60,000 (GBP)
Organisation Quorn Foods Limited 
Sector Private
Country United Kingdom
Start 06/2019 
End 06/2023
 
Description Mode of action of natural-product food preservatives
Amount £13,317 (GBP)
Organisation Wageningen University & Research 
Sector Academic/University
Country Netherlands
Start 08/2020 
End 11/2020
 
Description Newton Fund Researcher Links
Amount £60,000 (GBP)
Funding ID 2017-RLWK8-10388 
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 01/2019
 
Description Spoilage-yeast metabolism, reduced-sugar formulations and implications for food preservation
Amount £54,000 (GBP)
Organisation Lucozade Ribena Suntory 
Sector Private
Country United Kingdom
Start 12/2020 
End 11/2023
 
Description "Inoculum size matters: relationship between preservative MIC and heteroresistance in spoilage yeasts" : ICFM meeting, Utrecht, Netherlands 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact new contacts / collaborations
Year(s) Of Engagement Activity 2022
URL https://www.foodmycology.org/images/download/Abstract_book_ICFM_2022.pdf
 
Description "Preservative resistance of spoilage yeasts at low glucose and relevance for reduced-sugar formulations" : ICFM meeting, Utrecht, Netherlands 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact new contacts and collaborations
Year(s) Of Engagement Activity 2022
URL https://www.foodmycology.org/images/download/Abstract_book_ICFM_2022.pdf
 
Description BBSRC-FAPESP workshop Sao Paulo, Understanding and managing resistance, including novel methods, for pathogen and pest control 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact BBSRC funded participation in this workshop in Brazil, to establish new partnerships
Year(s) Of Engagement Activity 2017
 
Description Conference organiser 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference organiser, British Mycological Society Main Meeting
Year(s) Of Engagement Activity 2017
 
Description How to tackle problem fungi? new approaches from unlikely beginnings - UK Fungus Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A talk for "UK Fungus Day"
Increases awareness of fungi and their impacts among the general public incl policy makers
Year(s) Of Engagement Activity 2020
URL https://www.ukfungusday.co.uk/
 
Description Tackling fungal food spoilage and crop disease, Brazil relevance 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Supporting a new collaboration
Year(s) Of Engagement Activity 2017
 
Description Unilever visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Research collaboration presentation
Year(s) Of Engagement Activity 2016
 
Description Water resilience and food security in Ghana 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Intended to promote new research collaborations towards future funding opportunities, e.g., GCRF
Year(s) Of Engagement Activity 2018
 
Description Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I co-organised a UK-Brazil researcher links 3-day workshop on the fungal threat to food security, which took place at Univ. Sao Paulo in Sept 2018
Year(s) Of Engagement Activity 2019
URL https://www.britishcouncil.org.br/events/researcher-links-workshop-food-security