There's nothing worse than having a customer open a bottle of wine expecting pleasant aromas and instead smelling off-putting barnyard or dead mouse aromas. Because Brettanomyces bruxellensis (Brett) can ferment sugars and create unappealing flavor and aroma changes, winemakers design quality control programs to discover it early enough to correct the problem.

Winemakers use several methods to identify Brett in wines and differentiate it from Saccharomyces, including microbiological culture plating. While the low cost and the average lab technician's familiarity with inoculating a sample of wine onto a nutrient agar medium to culture microorganisms make it an easy choice, there are drawbacks.

"Current methods, such as microbial plating, take about seven to ten days for Brett to grow on a plate," says Daniel C. Gusmer, a program scientist in fermentation development at Gusmer Enterprises. "By that point, if you have a problem either in actively fermenting wine or in your barrel, it could be too late to intervene. Depending on the levels of Brett, that product could be lost."

Gusmer points out that another commonly used tool, quantitative polymerase chain reaction (qPCR), offers a faster turnaround but will amplify any DNA present without differentiating live cells from dead ones, which results in overestimation. However, qPCR's main pitfall is the complexity of its extraction, purification, and thermal amplification processes, which make the results prone to error. As a result, many wineries outsource lab testing of qPCR and its close cousin PCR to specialized laboratories, which incurs costs and lengthens the turnaround time, making the results less immediately actionable.

Searching for faster, reliable results

Winemaking experts have been discussing the value of flow cytometry as an alternative to these techniques for identifying Brett at conferences and in scientific papers for 10 or 15 years. They have pointed out how quickly and accurately flow cytometry can detect Brettanomyces bruxellensis (Brett) in wine samples. They've often called attention to its usefulness for testing wine while it is fermenting, aging in the barrel, or during quality control on bottling lines.

Gusmer has been using flow cytometry at the Gusmer Fermentation Center in Santa Rosa, CA, which includes its R&D department. Family-owned Gusmer Enterprises has served the wine industry with innovative products and services since its incorporation in 1924. Today, it is a leader in manufacturing and distributing filtration products, including depth filter material, fiber, sheets, stacks, and cross-flow.

"We're researching new solutions for customers," he explains. "We do internal work to support our product lines and validate new technology platforms for the wine industry. That is where we saw the need for a quick, accurate microbial detection system, and our research came across flow cytometry."

He was looking for a solution that:

A) Distinguishes between different species of yeast and bacteria, which automated cell counters don't do well;

B) Extremely fast, instead of waiting a week or ten days as for traditional microbial plating results;

C) Easy to use and thus cross-trainable for operators; and

D) Low margin of error and a low coefficient variable.

A better mousetrap

He found his solution in the CyFlow BrettCount by Sysmex Corporation, a pioneer in developing flow cytometers, which partnered with Vermicon, which specializes in fluorescent microscopy, to combine Vermicon's Vermicon Identification Technology (VIT) with Sysmex's flow cytometer.

"Most enological flow cytometry got its start in fluorescent microscopy," he notes. "Vermicon developed gene probes that attach to a particular portion of a yeast or bacteria's rRNA, a portion that also happens to degrade rapidly, almost instantaneously, when a cell dies. So, unlike qPCR or PCR, which will amplify any DNA, whether the cell is dead or alive, these gene probes will only attach to live cells. That is the inherent benefit."

Gusmer Enterprises acquired a CyFlow BrettCount detection system in 2021, six months after its release, and has used it in the Fermentation Center ever since. It is a fast and reliable solution for detecting Brett contaminations. A well-designed quality control testing program using CyFlow BrettCount allows Brett to be detected early enough to permit corrective actions that preserve the wine's sensory quality.

"We used the CyFlow BrettCount as a research tool with great success for about two years," says Gusmer. "Sysmex designed these flow cytometers and kits specifically for the beverage market, and we realized we could make a tool available to the wine industry that has not been accessible until now. We entered a distribution agreement with Sysmex to bring this platform to wineries and other beverage companies in July 2023."

For more information on acquiring these easy-to-use flow cytometers, contact gusmerwine@gusmerenterprises.com.