Project Summary
The global beer market was valued at £297 billion in 2017, and is projected to reach £688 billion by 2025. A significant proportion of this growth will be driven by developing markets such as Africa and South America. This growth puts increasing pressure on available raw materials and greater demands on the hygiene of brewery plants. The industry is going to face increasing pressure to ensure the food safety of their products. This is further necessitated by the growth of craft breweries, who are less skilled in hygiene and food safety, and the rise in popularity of low and no-alcohol beers which have a greater risk of spoilage.
Spoilage of ingredients or in-process products usually only leads to a direct financial loss, the consequences are more severe if it concerns a final product which has reached the market. On top of the costs of lost product and its recall, customer confidence and brand image may be seriously affected. To avoid this, new techniques are needed to optimise quality assurance (QA) in manufacturing plants to ensure the food safety and quality of products. These new techniques should support rapid identification of microorganisms in the raw materials and final product.
This project will investigate the potential of a new platform based on aptamers to provide fast, reliable and sensitive identification and quantification of spoilage organisms in brewery samples (wort, beer, CIP). Currently there are no methods available to the industry that allow rapid direct detection and identification of spoilage organisms. This project will provide the technology for the next generation of QA tools.
Aptamers are short oligonucleotides, either RNA or DNA, which have been successfully used to specifically target a range of different cell types or molecules. The nucleotide sequence of the aptamers causes it to fold into a specific three dimensional shape, which allows it to bind to specific targets. As such, aptamers are similar to antibodies, but possess key advantages including size, increased stability, reduce immunogenicity and simple conjugation chemistry. Aptamers can be used in conjunction with a sensor to produce aptasensors which enable detection of a designated target through the measurement of changes in optical, electrochemical or mass properties. Aptamers are selected from a random library of oligonucleotides through a process of systematic evolution of ligands by exponential enrichment (SELEX).
The key outcomes of the project:
1. Selection and characterisation of aptamers for key brewing spoilage microorganisms
2. Cloning and sequencing of aptamers
3. Use of flow cytometer to assess the binding ability and specificity of aptamer to target microorganism
4. Assessment of isolated aptamers for their effectiveness for detection of spoilage organisms in industrial samples
Full Project Description
The student will be trained in general laboratory techniques, including working with yeast and bacteria cultures.
The student will aim to complete 3 tasks for the project:
1. Selection and characterisation of aptamers for beverage spoilage microorganisms using a cell-SELEX procedure. Following negative selection on a culture yeast, the aptamer library will be incubated with spoilage bacteria (Lactobacillus) / yeast (Brettanomyces) to select aptamers that specifically recognise the target cell. Multiple rounds of cell-SELEX (a minimum of 5) will be completed. Negative selection will be performed against S. cerevisiae the key organism present in beverage fermentations.
2. Cloning of isolated aptamers into Escherichia coli. Positive clones will be sequenced to identify individual aptamer sequences. Aptamer sequences will be analysed by a number of open access software programmes to determine sequence similarity and to predict secondary structures, which are important for aptamer binding.
3. Visualisation of the aptamer with a fluorescent tag binding to the target organism in a mixed culture