April 2015

Wine, Cheese, and the Microbiomes That Influence
Their Flavor
Researchers use the MiSeq® System to identify the microbiome profiles of wine and cheese, andthe facilities in which they are made.
Introduction

A Different Wine and Cheese Pairing

Most microbiology studies focus on how to identify and remove
microbes from our food—and for good reason. Pathogens like
Salmonella enterica, Listeria monocytogenes, and Escherichia coli
sicken countless people every year. But University of California, Davis(UC Davis) food scientist David Mills, PhD, is quick to point out thatmicrobes are actually the true chefs behind some of our favorite foods.
Lactic acid bacteria metabolize the lactose in milk, helping convert
it into the savory cheeses that we enjoy such as cheddar, brie, or
pecorino romano. And the yeast Saccharomyces cerevisiae performs
its magic on grapes, transforming their juice into a seemingly infinite
variety of wines for every taste and occasion.

Dr. Mills began his career in biochemistry and biotechnology. Asa PhD student at the University of Minnesota, he switched to themicrobiology program after becoming interested in the work of Larry
McKay, PhD, a microbiologist who studies the bacteria that makecheese. This appealed to Dr. Mills’ interest in translational science.
“While I enjoy basic research, what drives me is studying its applicationin processes and the environment,” Dr. Mills stated.

Dr. Mills studies the microbiomes of cheese and wine, and the facilities
that produce them, to understand how microbes work their magic.
It’s a question that scientists have only recently been able to tackle,
thanks to next-generation sequencing (NGS) instruments, like theMiSeq System.

He decided to focus on the genetics of Lactococcus lactis, one of
the lactic acid bacteria needed to make cheddar cheese. After ashort postdoc at North Carolina State University, Dr. Mills was hiredby the renowned Department of Viticulture and Enology at UC Davis.
Although wine’s main fermenter is S. cerevisiae, lactic acid bacteria
also perform some of the fermentation. It was a wine and cheesepairing of a different sort, and Dr. Mills loved it.

Sampling Cheesemaking Facility Microbiomes

He hopes to discover how cheeses and wines cultivated in different
regions develop their own distinctive flavors by observing andcataloging their unique constellation of microbes. “The concept ofregionality in food might be traceable through the microbes that areassociated with it,” Dr. Mills said. “The microbiota profile at the front
end, before the grapes are fermented, could correlate with the profileof the chemical constituents of the finished wine. A very exciting
aspect of our research is that we might be able to predict flavor
outcomes by studying these microbes.”

Understanding the microbial ecology of any food involves more thana focused study of the hundreds or thousands of different microbes
found in the food itself. It also means studying the microbes found in
the facility where the food is prepared and packaged.

David Mills, PhD, is a Professor in the Departments of Food Science andTechnology, and Viticulture and Enology at the UC Davis.

UC Davis viticulture and enology student Javier Avina prepares a fermentercontaining crushed grapes for primary fermentation.

“We’re very interested in understanding the microbial landscape of
the whole food production facility, such as the seasonality of microbial
movement inside the facility,” Dr. Mills said. “For example, howactivities inside the facility lead to microbial transfer back and forth.

Photo courtesy of UC Davis Communications

For Research Use Only. Not for use in diagnostic procedures.

April 2015
Often, I’m studying facilities where microbes are purposely introduced
for making cheese or wine. The question is, do these microbialcolonies grow to populate the entire place? Where else do they end
up? In instances where sanitation is performed correctly, we want tounderstand how other microbes present in the facility take over.”amplicon sequences tell the researchers which microbial species arepresent in a given sample, allowing them to take a census of microbes
across a facility and over time.

No matter the type of facility being studied, identifying these microbes
means taking samples—lots of them. Dr. Mills and colleagues use
cotton swabs to take thousands of samples from buildings and thefoods they produce.

Back at the UC Davis winery, Dr. Mills and Bokulich sampled thewhole facility about 150 times using cotton swabs at several points
throughout the winemaking season. They started in August, before
the grapes were harvested and brought inside. They returned in themiddle of the season, as the grapes were brought into the winery to be
crushed. And they made a final visit in mid-December, when the facility
was being cleaned and the winemaking equipment stored for theseason. As they predicted, they saw dramatic shifts in the microbes
over that time. They also found some things they didn’t expect to see.
For example, S. cerevisiae arrived on the doorstep of the winery long
before a single grape came in, perhaps brought in by workers walking
back and forth from the vineyard to the winery.2

After chemically isolating and extracting the DNA, Dr. Mills originallyemployed “very rudimentary tools” to identify the different microbes
by hand, using denaturing gradient gel electrophoresis. “Old schoolmethods had very low throughput,” Dr. Mills said. “We could only
analyze 20 samples at a time and the resolution was terrible. Graduate
students hated it because it was a tough method and very timeintensive. We could get an indication that there were a bunch ofmicrobes present, but we weren’t sure what they were.”

Role of House Microbiota in Cheese and Winemaking
While the interest in decoding the microbes that make wine, cheese,and beer has always been high, the research stalled in the absence ofefficient tools. This changed several years ago, when the sequencing
core at UC Davis invested in NGS and purchased several MiSeq
Systems. Taking advantage of this advanced technology, Dr. Mills
began identifying microbes from large numbers of samples. Together
with graduate student Nickolas Bokulich, he began to look moreclosely at the microbes in a California artisanal creamery to see if
a facility-specific or “house microbiota” could be identified. They
sampled microbes from the surfaces of the washed rind cheeses (aclass of cheese that includes Limburger and other pungent varieties),and examined some of the surfaces in the facility itself.1“We could see that the microorganisms on the surface of a cheese
were the same ones present on the surface of some parts of thefacility,” Dr. Mills stated. “We’re still unsure which direction themicrobes colonized: from the cheese to the facility surfaces, from thesurfaces to the cheese, or in both directions over time.”
Regardless of the colonization dynamics, the results supported theidea that a house microbiota helps to inoculate a cheese or wine andcause it to take on a specific flavor. Vintners in the Bordeaux region ofFrance have said for years that the winery itself inoculates the wine. It’sa concept called terroir, the idea that the place where a wine or food isgrown or made is ultimately expressed in its final taste.
“People will taste a wine and say ‘that’s a Bordeaux,’” Dr. Mills said. “Agood sommelier can even tell you the vintage and region of origin. Yet,
we don’t know what causes that regionality. How do we connect thesense of place to actual sensory metabolites in the wine, so we canunderstand why a Napa Valley cabernet delivers a specific flavor that
can’t be found in a cabernet from the California Central Valley?”
No one knows exactly what creates terroir in a food, but Dr. Mills’ workis showing that microbes likely play a role. His sequencing analysis
studies begin with extracting DNA from samples and amplifying
portions of bacterial and fungal ribosomal genes with different setsof barcoded primers, some of which were developed in his lab. The
library sets are sent to the UC Davis Genome Center, where theMiSeq System is used to sequence 500 samples in a single run. The

For Research Use Only. Not for use in diagnostic procedures.

Identifying Winery Microbiomes

Dr. Mills and his colleagues expanded their study to wineries
throughout California, looking at the microbes that grew on grapes andin the facilities over 1 to 2 seasons. The goal was to see if microbial
populations were unique for different wines from different regions,and to study how these microbes moved through the system fromharvesting to the final product. They also sought to document the roleof microbes in producing a specific wine and its flavor.
Clearly, the flavor molecules in the grapes themselves strongly
influence the final flavor of the wine, and certain regions produce
grapes that yield better wines than others. “Whether microbes play
a 1% or an 80% part of the flavor-generation process, I don’t know,”
Dr. Mills stated. “The Holy Grail in wine is to understand where a goodwine flavor comes from, and we know that there is an amalgam of
microbes needed to produce the final product. Many of the mostdesired wines have much more complex flavors that are driven bymore than just S. cerevisiae. We’re attempting to understand theconstellation of microbes that delivers that actual flavor.”
The research showed that “there are different sets of microbes
entering the wineries from different regions on the same grapes,”
Dr. Mills said. “We know some of those microbes influence the flavorof wine, providing the potential connection between something in thevineyard to a flavor molecule in the wine.”
Dr. Mills and his team studied must, the freshly pressed juice createdfrom crushed grapes before they are fermented.3 The data revealed
that the microbes entering a winery on grape must are region-specific.
For example, the microbes on chardonnay must from the California
Central Coast are different from those on chardonnay must from Napa
Valley or Sonoma County.
“If you take the same vine type and grow it in Sonoma and in Napa,
I wouldn’t expect to see that much difference in the microbial
landscape,” Dr. Mills stated. “Plants, like humans, have typicalmicrobes associated with them. So I was a bit surprised at how
robustly we could differentiate the regions based on the microbialcommunities we found.”Different populations of microbes that enter into fermentation could
cause flavor differences in the final product, a hypothesis that Dr. Millsand other scientists are just beginning to test.

April 2015
NGS Enables Efficient, High-Quality Microbiome Research
None of this work would have been possible without NGS systems,
such as the MiSeq System, and bioinformatics programs that help
researchers make sense of the data. “The irony of the situation is thatcollecting the samples is now the biggest problem I have in performing
these studies,” Dr. Mills said. “Processing the samples and runningthem on a sequencer is easier than going to a facility and spending
8 hours obtaining 300 swabs, all documented so we know exactly
where the sample was taken. In one of the papers we’re submitting
soon, the winery had to collect about 2000 fermentation samples forus. That’s a large number of samples and they all have to be labeledcorrectly and stored somewhere. It’s a lot of work.”
For Dr. Mills’ research, the MiSeq System provides an optimum
combination of speed and cost. “I like the MiSeq System because
it’s a scaled down version of a high-throughput sequencer,” Dr. Mills
stated. “It turns around samples quickly and I know that the quality
is good. While I’d love to see a larger read, I’m happy with the reads
that I get.”His work has implications beyond identifying the microscopic creaturesthat help to make some of our favorite foods and wine. It might also
help us understand how microbes move through our world.
“Our goal is to develop a microbial map of a whole facility,” Dr. Mills
added. “One of the beauties of using NGS tools is that we canprocess a large number of samples. Our MiSeq runs are often 500
samples or more. In the old days, plating 500 samples would nothave been feasible. We could never have performed the kind of highthroughput studies that we can today. With NGS data, we are opening
up a completely new window, enabling us to view the microbial
landscape of food facilities.”

References
1.

2.
3.

Bokulich NA and Mills DA. Facility-specific “house” microbiome drives
microbial landscapes of artisan cheesemaking plants. Appl Environ
Microbiol. 2013;79(17): 5214–5123.
Bokulich NA, Ohta M, Richardson PM, Mills DA. Monitoring seasonalchanges in winery-resident microbiota. PloS One. 2013;8(6):e66437.
Bokulich NA, Thorngate JH, Richardson PM, Mills DA. Microbial
biogeography of wine grapes is conditioned by cultivar, vintage, and climate.
Proc Natl Acad Sci USA. 2014;111(1):139–148.

Learn more about the Illumina system mentioned in thisarticle and the art of cheesemaking:••

MiSeq System, www.illumina.com/systems/miseq.html
Illumina SciMon Video: Cheesemaking: Ancient Art or the Simplicityof Science, www.youtube.com/watch?v=M0QUzpRCvP4&feature=
youtu.be&list=PLKRu7cmBQlahAeoVCcb-pM1oVCKFkggFh

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April 2015Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
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© 2015 Illumina, Inc. All rights reserved. Illumina, MiSeq, and the pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s)in the U.S. and/or other countries. Pub. No. 1370-2015-001 Current as of 14 May 2015