Molecular Methods for Microbial Analysis in the Food Industry

Steve Flint, School of Food and Nutrition, Massey University, Palmerton North, New Zealand
Ó 2016 Elsevier Inc. All rights reserved.

Molecular biology has moved rapidly over the past 20 years. Medical research has lead the way in the development of techniques
that have been picked up progressively by the food industry for the identification, characterization, and source attribution studies
for microorganisms involved in the manufacture of fermented foods or those that threaten the safety and quality of foods. Today
a PCR machine for DNA amplification and identification of microorganisms is common in all food microbiology teaching, research,
and many contract testing laboratories. We can now identify nonviable and nonculturable microorganisms, that is, those that we
cannot grow in the laboratory. We know microorganisms are present even though we do not have the ability to culture them. This is
revealing new information about the microbial world.
Apart from the use of sophisticated cloning and expensive sequencing work in some of the research laboratories, occasionally
used for food research, the earliest techniques to be used by the food industry focused on the detection of pathogenic bacteria using
test kits that relied on matching DNA through DNA hybridization. These have now largely been replaced by the species specific
PCR-based detection kits which are very efficient at rapidly confirming the identification of pathogens such as Listeria and
Salmonella. These test kits come in a variety of formats including the semiquantitative ‘real-time PCR’ assays. These techniques
still require the enrichment of low numbers of bacteria in food to produce the numbers needed to enable identification;
however, the main advantage is the simplicity and speed of confirming the presence of a particular bacterium. There is an added
cost compared with the traditional methods for microbial identification. However, these methods are now widely used in the
contract testing laboratories for pathogen detection.
Techniques used for ‘fingerprinting’ different bacteria from the same species have been available for some time, starting with
some very basic tests that cut DNA into segments that are specific to a narrow group, providing a bar code–like pattern that could
trace the origin of a contaminant if samples are taken from a number of potential sources (Giraffa and Carminati, 2008). A variety of
new molecular fingerprinting techniques based on the PCR amplification of specific sequences of DNA are now more widely used
for microbial tracking. In food research, these have been useful in determining the origin of food contaminants (Fu and Li, 2015).
The most recent development is the use of next generation sequencing (NGS) (Mayo et al., 2014). This is a rapid method for DNA
sequencing that allows the sequencing of the whole genome of the microorganism. This can then be compared with databases that are
rapidly growing in their sophistication with some very specific information available to trace microorganisms. This new technology
promises to revolutionize the ease, efficiency, and accuracy of tracing the source of microbial contaminants and determining the
changing populations of microorganisms in foods under different manufacture and storage conditions. NGS has already found novel
microorganisms in different foods and food processes (Mayo et al., 2014). This is likely to assist in improving the quality and safety of
foods and in optimizing food fermentation, maturation, and preservation. Some of the microorganisms that NGS is detecting in
foods could explain variations in the sensory attributes and functionality of different batches of food, in particular food from different
origins. One of the most exciting developments we expect from NGS is the identification of new starter microorganisms leading to
new fermented foods. In our laboratory, we are using NGS to understand variability between strains of bacteria that we formally
believed to be identical. This explains some of the phenotypic observations in biofilm formation and spore formation of these
bacteria and also shows how these bacteria have become adapted to a particular food niche. This can have implications in terms
of the effect on product quality. This is the most exciting development in food microbiology for many years. As the costs of NGS
reduces and the databases that NGS is producing increase, this tool will have a major impact on the future of the food industry.
Molecular techniques have a growing impact on detecting food authenticity. This has become necessary with reports of
adulterated food, such as the horse-meat scandal in the UK in 2013. Here horse meat was found in beef burgers, creating inter-
national headlines. Many of the techniques, such as species-specific PCR and RFLP techniques, used in microbial identification,
can be used to identify a specific contaminant in a food and are nicely reviewed by Singh et al. (2014). For example, if you
suspect horse meat in a beef product, you can use targeted PCR techniques to confirm that suspicion. However, if you want
to confirm authenticity of a product without any prior suspicion of potential contaminants, then a more robust screening tech-
nique is required. Once again, NGS provides the solution, enabling profiling of the product to confirm its composition. This
can be used for a whole variety of biological ingredients, including confirming the authenticity of fruit and plant material in
food (Ortola-Vidal et al., 2007).
As the costs of NGS reduces and the databases that NGS is producing increase, this tool will have a major impact on the future of
the food industry and will largely replace the myriad of other techniques, molecular and nonmolecular, that have been used until
now for both microbial identification and food authenticity.

References

Fu, L.-L., Li, Jian-Rong, 2015. Microbial source tracking: a tool for identifying sources of microbial contamination in the food chain. Crit. Rev. Food Sci. Nutr. 54 (6), 699–707.
Giraffa, G., Carminati, D., 2008. Molecular techniques in food fermentation: principles and applications. In: Cocolin, L., Encolini, D. (Eds.), Molecualr Techniques in the Microbial
Ecology of Fermented Foods. Springer.

Reference Module in Food Sciences http://dx.doi.org/10.1016/B978-0-08-100596-5.03397-7 1

2 Molecular Methods for Microbial Analysis in the Food Industry

Mayo, B., Rachid, C.T.C.C., Alegria, A., Leite, A.M.O., Piexto, R.S., Delgado, S., 2014. Impact of next generation sequencing techniques in food microbiology. Curr. Genom. 15,
293–309.
Ortola-Vidal, A., Schnerr, H., Rojmyr, M., Lysholm, F., Knight, A., 2007. Quantitative identification of plant genera in food products using PCR and pyrosequencing™ Technology.
Food Control 18, 921–927.
Singh, V.P., Pathak, V., Nayak, N.K., Verma, A.K., Umaraw, P., 2014. Recent developments in meat species speciation – a review. J. Livest. Sci. 5, 49–64.