Undergraduate Student Report 2022

Implications of reduced sugar for yeast physiology and potential for drinks spoilage

Jack Nichols, University of Nottingham

I am a Biotechnology student soon to begin the third year of my course at the University of Nottingham. With funding from the British Mycological Society, for which I am very thankful, I was able to carry out a 10-week project during the Summer of 2022. This was under the supervision of Dr Harry Harvey and Professor Simon Avery at the University of Nottingham’s School of Life Sciences.

My project was to investigate whether sensitivity of a key food-spoilage yeast to weak acid preservatives is affected by glucose concentration. This is important because of the drive to lower sugar content of foods, including soft drinks, by consumers and government regulations, without compromising food safety or shelf-life.

I worked principally with the spoilage yeast Brettanomyces bruxellensis, using a strain isolated from a reduced sugar (<5% w/v) soft drinks formulation. I compared preservative resistance of this yeast at low versus high glucose with that of the baker’s (and spoilage) yeast Saccharomyces cerevisiae.

Initially, I tested B. bruxellensis against a range of weak acids including sorbic and acetic acids using a 96-well microplate format. This format allowed higher throughput testing of the relative resistance phenotypes. To support these data, a checkerboard-style assay was adapted to be able to see how resistance to sorbic acid (the principal weak acid preservative used in soft drinks) varied over a range of glucose concentrations. Many yeasts are known preferentially to ferment at high glucose, but increasingly to switch to respiration as glucose is lowered. The comparison between the two yeast species was striking, as previous evidence indicates sorbic acid hypersensitivity of S. cerevisiae in the post-diauxic (respiratory) late growth phase (https://doi.org/10.1128/mSphere.00273-20), whereas this effect was not evident in B. bruxellensis (Figure 1). Further testing in McCartney bottles to measure fermentation according to evolution of gas pressure revealed that B. bruxellensis ferments relatively poorly. This evidence, in addition to microplate data showing that this yeast grows comparatively well in low glucose conditions, led to the idea that B. bruxellensis has a relatively robust respiratory metabolism. This could help explain why it was isolated as spoilage organism in soft drinks containing sorbic acid.

Figure 1

Figure 1. Growth curves showing growth (OD600) of B. bruxellensis in the indicated conditions over time. Low or high glucose = 0.1% or 2.0% (w/v).

 

 

 

 

Following this, I used confocal microscopy and flow cytometry to examine cells labelled with a fluorescent probe that reports on mitochondrial membrane potential. The results from these assays supported the idea that the respiratory state of B. bruxellensis may be more active and resilient to sorbic acid stress than in S. cerevisiae. Further investigation of these initial findings from my project is now planned.

Figure 2

 

Figure 2. Me analysing flow cytometry data  (background view: beautiful Nottingham)

 

 

 

 

The project also gave me the opportunity to join a visit to the Lucozade Ribena Suntory (LRS) site in Coleford, UK, as LRS is an industry collaborator of my host research group. This industry experience helped me learn more about how the laboratory research can be translated to the real world and the exciting potential for impact that research like mine has. I gained valuable insights to the industry activity and enjoyed being given some of their drinks to bring home with me!

I would like to thank the BMS and the Nottingham research group again for enabling this amazing opportunity. The chance to learn a range of skills that cannot be taught in the lecture hall and invaluable practical and industry experience, particularly given the limitations last year due to COVID, has been so rewarding. The skills that I have learned provide me with laboratory proficiencies that will help accelerate my learning and prepare me for my final year of studies and future career.

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Jack Nichols was supervised by Prof Simon Avery and Dr Harry Harvey, University of Nottingham.