Gregory Siragusa, Eurofins
Food Genomics

What’s in a Name? Probiotic Analysis and Genomics

By Gregory Siragusa, Douglas Marshall, Ph.D.
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Gregory Siragusa, Eurofins

In the era of newer probiotic organisms, asking the right questions is crucial.

In short, in the world of regulatory and probiotic microbiology the “name” is critical. Whether you are a probiotics manufacturer, blender or user, we are all likely aware that usage and sales of probiotic strains of bacteria and yeasts is burgeoning. Estimates of sales growth are impressive, with $24 billion and $5 billion in human and animal markets respectively projected by the year 2024.1,2Although the organisms approved as human and animal probiotic are a huge list, it is quite large and varied. Prove it to yourself by visiting your local grocery store or pharmacy and take a trip down the aisle where probiotic supplements are displayed. Read content labels and see if you recognize the microbe names. There are many probiotic organisms’ names that you are likely to be familiar with, most of which are lactic acid bacteria (LAB). However, we are quickly entering an age of more novel or even new probiotic organisms that may be unfamiliar to you. Some of which are not always as easy to culture as the LAB.3 On the same labels you may see claims of viability and cell population declarations (usually in CFU’s or colony forming units). Also, many probiotics are retailed in dry form, while others are marketed as liquids. As food safety scientists and practitioners, questions are probably popping into your head as to how probiotic species and populations are verified and how these various preparations survive expected shelf life.

Most will agree that before anyone starts consuming pills or eating foods with billions of viable bacteria, it is obviously a prudent idea that the manufacturer has the means to assure safety and quality. The details and scope of probiotic safety and microbial analysis are much too complex and broad to deal with in a few pages. For more details we direct the reader to two key publications.4,5

Identity, viability and populations are attributes largely measurable by methods that rely on culture, phenotypic analysis, genomics and combinations thereof. Here we will share a primer on genomic methods for probiotic analysis starting with a very basic aspect critical to all of microbiology—taxonomy and asking the “right” questions.

Why Not Just Perform a Plate Count? Revisiting Taxonomy

The process of identifying or classifying organisms, also known as the science of taxonomy or systematics, has sometimes been given less-than-stellar treatment among the community of microbiologists. We are frustrated when taxonomists change genus or species names just as people learned the old existing names. But every dog will have its day, and for microbial systematists, that day has arrived since application of genomic tools to the taxonomist toolbox has coincided with growth of the probiotics industry. Practically speaking, for the probiotic microbiologist, there is a lot more to a name than just nomenclature. Microbial taxonomy, and specifically bacterial taxonomy, becomes vitally important as more and more products are produced and as regulations increase in scope. Bacterial nomenclature is an ever-changing field, but at least naming has become more centralized with its own website.6

For the probiotic manufacturer, some important questions require answers: “Is it ‘my’ strain?”, “What’s in the mixture?”, “Is the label accurate?”, and “Are they alive?”. So why are we addressing this topic as a subject for Food Genomics? Confronted with the shear variety of bacterial types, it is easy to see why and how genomic tools offer a solution to this complexity. We now have tools that augment, complement, or even in some cases, replace cultural microbiology as a means to classify, identify and analyze probiotics (see Table I).7

 General Method Application Notes
 Targeted microbiome Genus/Species level resolution
Bacterial or fungal profiles (16S/ITS gene)
Suitable for multi-strain probiotic products
Unknown or QA analysis
 Shotgun metagenome Species/Possible Strain level resolution
Bacterial and fungal profiles in same assay
Well suited for multi-strain probiotic products
 Qualitative microarray Species/Possible Strain level resolution
Non-quantitative, descriptive only
Well suited for multi-strain probiotic products
 PCR Species/Strain, probe designed specificity
Qualitative
 qPCR Species/strain probe designed specificity
Quantitative against CFU standard curve
Can be designed to detect viability
 Flow Cytometry
(Gene Probe-Based)
Probe designed specificity
Quantitative against CFU standard curve
Viable, injured, dead cell detection possible
High throughput
Table I. Genomic Tools for Probiotic Analysis

“Why not just perform a plate count?”. Obviously plate counts have a pivotal role in the analytical microbiology of probiotics and will likely remain a gold standard for enumeration of viable counts. In fact, the unit of viable cell counts, CFU is the recommendation for use to verify probiotic populations.8 Unfortunately, most plate count methods do not name or identify the microbes we count as colonies. Occasionally, with precise selective and differential media the identity of the colonies growing on the plate can be reliably called, but misidentification is common. Other tools, such as PCR, are the tool of choice for amplifying specific genes from an organism’s DNA. Quantitative PCR (qPCR) and flow cytometry both rely on a probe specific for a species or even a strain in order to estimate cell numbers, including viable cell counts.7, 9,10,11, 12

So how will modern genomics help you with the analysis of your probiotics? The following are some questions, examples and comments that illustrate genomic applications for probiotic analysis that you should be familiar with. These methods, whether sequencing-based, PCR-based or flow cytometry-based (using gene probes not antibodies), all require some form of sequence determination, detection/hybridization and analysis.

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About The Author

Gregory Siragusa, Eurofins

About The Author

Douglas Marshall, Ph.D., Eurofins
Douglas Marshall, Ph.D.
Chief Scientific Officer

Douglas Marshall is chief scientific officer with Eurofins Microbiology Laboratories, Inc., a division of the global life sciences company Eurofins Scientific. He also is cofounder and director of the Food Safety Institute, LLC, an integrated consulting and analytical services company affiliated with Eurofins. He currently holds adjunct professor positions with Colorado State University and Florida State College. His former positions include associate dean and professor of public health at the College of Natural and Health Sciences, University of Northern Colorado; adjunct professor with the Colorado School of Public Health; professor of food science, nutrition, and health promotion at Mississippi State University; assistant professor of food science at Louisiana State University; contributing editor for the peer-reviewed scientific journal Food Microbiology, and four consecutive terms on the editorial board of the Journal of Food Protection. He is a frequent consultant to NIH, WHO, FAO, USDA and other government agencies and private companies. His research and expertise has been featured in popular press venues such as Consumer’s Reports, Fine Cooking, USA Today, Fitness, Health, Men’s Health, Chemtech, Nature Science Updates, and ASM Journal Highlights. Marshall is a frequently invited speaker and a prolific book chapter writer. With more than 250 publications and more than 150 invited presentations, his scientific research and outreach interests focus on improving the microbiological quality and safety of foods. Among these was the completion of the four-volume Handbook of Food Science, Technology, and Engineering, which he co-edited. He has been the recipient of a number of awards for his scholarly efforts including the Mississippi Chemical Corporation Award of Excellence for Outstanding Work and the International Association for Food Protection Educator Award. He is a fellow of the Institute of Food Technologists, where he has previously served as chair of two divisions and two regional sections, member of the board of directors, an Inaugural member and chair of the International Food Science Certification Commission, and a founding member of the Global Traceability Center.

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