I would venture to say that most people who enjoy a bottle of wine never think of all the steps and considerations involved in bringing that beverage to them for their pleasure. Winemaking has been an endeavor for thousands of years and is both an art and a science.
To determine what’s involved with regard to the science—specifically the analytical aspects—of winemaking, I spoke with representatives of a well-known university enology department, the largest wine company in the world, and the largest independent laboratory specializing in wine analysis.
Analyses During Winemaking
Winemakers analyze for different components in grape juice to assess their levels to determine harvest date. They measure the nutrient status of the incoming juice and augment it with a nitrogen source if necessary. The subsequent winemaking steps involve letting the skins soak in the juice to develop a red color for red wine or removing the skins and seeds for white wine, allowing the juice to ferment, aging the wine, generally in oak barrels, then clarifying and stabilizing the wine before bottling, after which it may be further aged. Sulfur dioxide is often added to the juice before fermentation to minimize oxidation and control the growth of spoilage organisms.
Every wine undergoes a primary fermentation, during which yeast—primarily Saccharomyces cerevisiae— converts most of the sugars in the grape juice into ethanol. A secondary fermentation is used to produce sparkling wines, where added yeast ferments added sugar to produce a bit more ethanol and the carbon dioxide necessary for the bubbles. Some wines undergo a second bacterial “fermentation,” in which lactic acid bacteria convert malic acid into lactic acid, softening the mouthfeel of the wine and also producing other flavor and aroma products.
At each of the stages in winemaking, analytical tests are conducted. Common tests include °Brix (a measure of the total soluble solids in the juice, primarily the sugar content), pH, titratable acidity, residual sugar, free SO2, total SO2, volatile acidity (basically a measure of acetic acid), and percent alcohol. Susan Ebeler ([email protected]), Professor in the Dept. of Viticulture & Enology at the University of California, Davis, said that pH and titratable acidity are commonly measured by titration, generally using autotitrators; °Brix by refractometers; soluble solids by density measurements using hydrometers or oscillating quartz microbalances; glucose and fructose (or reducing sugars) by enzymatic analysis; volatile acidity by distillation or enzymatic analysis; SO2 by redox titration; and nitrogen by ammonia ion electrode.
Ebeler said that wine analysis is complicated by the many types and styles of wines being produced—red, white, rose, dry, sweet, sparkling, high-alcohol, low-alcohol, etc.—and that advances are being sought in speed and ease of use of analytical methods and instruments. There is a lot of interest in rapid and automated technologies, she said, such as sensor-based technologies and near-infrared and ultraviolet-visible spectrometry. Electronic noses and tongues, involving polymer sensors and mass spectrometry, constitute a rapid method for profiling volatiles or nonvolatiles in wine but are not being used much yet, she said.
All these methods, she added, rely on databases and chemometrics to classify samples. Foss North America’s WineScan™ (www.foss.us) and Anton Paar’s Alcolyzer Wine (www.anton-paar.com) are examples of commercial instruments that use NIR spectroscopy for automated analyses (see sidebar on this page).
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Jack Thorngate ([email protected]), Applications Chemist, R&D, Constellation Wines U.S., St. Helena, Calif., said that the instrumentation used for the analyses varies with the size and resources of the wine company. For pH, small companies may conduct titration with burettes, while large companies use autotitrators. For ethanol, small companies may use ebulliometers, measuring boiling point suppression, while large companies use NIR spectrophotometers. For SO2, small companies may do iodine titration, while large companies use automated instruments such as Foss North America’s FIAstar. According to Thorngate, automation reduces manpower costs and improves accuracy, precision, and speed.
In addition to the analyses mentioned above, other tests may be conducted, he said. For example, for red wines, winemakers analyze for phenolics and flavonoids, including anthocyanins and tannins. Analysis of specific flavonoids may require a liquid chromatograph-mass spectrometer. For tannins, methylcellulose precipitation or protein precipitation methods may be used. Protein precipitation is correlated with human perception of astringency. Flavor analysis requires a gas chromatograph-mass spectrometer, the most sophisticated of which is a 2-dimensional gas chromatograph coupled with a time-of-flight mass spectrometer. There’s still no convenient way to rapidly analyze flavor except by human sensory analysis, he said, so many wineries analyze for specific defects, such as sulfur defects, pyrazine defects, etc., instead.
The largest wineries, such as E&J Gallo and Constellation have in-house R&D groups heavily invested in instruments such as LC-MS and GC-MS to help them understand flavor chemistry, he said. These and other companies want to be the best they can be while keeping costs within control and generating the profit they need to go forward.
Gordon Burns ([email protected]), President & Technical Director, ETS Laboratories, St. Helena, Calif., said that analyses begin in the vineyard, with questions relating to grape maturity. A juice panel analysis is conducted, including measurement of the fermentable sugars glucose and fructose; acid balance, specifically tartaric and malic acids; potassium, important in buffer capacity; titratable acidity; and pH, which influences color, cold stability, heat stability, and phenolics content. Burns said conversion of malic acid to lactic acid by malolactic bacteria during or after fermentation may impart desirable sensory characteristics and stability. The bacteria can produce a myriad of chemicals, such as diacetyl, a valued character in some wines but undesirable in others.
During fermentation, fermentable sugar levels are the primary component monitored, he said, unless the fermentation slows or exhibits problems. Most S. cerevisiae strains are glucophilic and prefer glucose to fructose. If the yeast is stressed and the primary sugar left in the fermentation is fructose, the fermentation can slow or stop. If that occurs, the winemakers will monitor fermentations for organisms that might be interfering with fermentation or causing problems with the wine. At the end of fermentation, glucose and fructose should be at trace levels if the goal is a dry wine. If the winemakers want a sweet wine, they may stop the fermentation sooner.
The bulk of the analyses, he said, are conducted during aging, primarily to prevent wine from microbial degradation during storage. Winemakers monitor the level of SO2 and check for the presence of spoilage yeasts and spoilage bacteria.
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Detecting Spoilage Organisms
Burns said that despite the best winemaking practices, microbial contamination often occurs during wine production. Spoilage microbes are capable of survival and growth in the wine, potentially producing off-flavors, off-aromas, and turbidity. Microbiological contamination is often undetected until these problems become noticeable by sensory evaluation.
The use of polymerase chain reaction (PCR) is becoming more widespread in the food industry for identifying and quantifying foodborne pathogens, Burns said, and about six years ago ETS Laboratories began development of PCR-based tools to identify and quantify spoilage organisms that affect wine. The company developed a system based on the Scorpions™ PCR technique that can be used to test samples simultaneously for a number of yeasts and bacteria that cause wines to spoil.
The spoilage yeasts include Brettanomyces, which can produce volatile phenols partially responsible for undesirable aromas and off-flavors, and Zygosaccharomyces, which can cause turbidity and carbon dioxide in bottled wines. The spoilage bacteria include Pediococcus, which can cause undesirable changes in wine texture and generate biogenic amines; Lactobacillus, which can produce high concentrations of diacetyl, which often causes undesired buttery flavors, and can also produce excessive amounts of acetic acid in a short period of time, often during sluggish or stuck fermentations; and Gluconobacter and Acetobacter, which can produce acetic acid in the absence of SO2 and presence of oxygen. The latter two bacteria can also cause elevated volatile acidity in wines exposed to air, and their presence indicates that wine conditions may support growth of other spoilage bacteria and yeasts.
The Scorpions-based system isolates the microorganisms from the wine sample, lyses the bacterial and yeast cells simultaneously to release their DNA, then extracts, amplifies, and hybridizes the DNA automatically for subsequent identification and quantification. Advanced robotics are used for a number of steps in the DNA extraction and reaction setup process, and real-time thermo-cyclers from Roche and Qiagen are used for the analysis. Scorpions panels accurately quantify the total number of viable cells in a sample. The system effectively cuts turnaround time to one day or less, Burns said, allowing winemakers to take remedial action before spoilage occurs.
The Scorpions platform was developed by the UK medical diagnostics company DxS Ltd. (www.dxs.com) and licensed exclusively to ETS Laboratories for wine analyses and to other companies for other purposes, including DuPont Qualicon for food safety analyses. Burns said that genetic analysis has advantages over traditional plating methods in that it can provide results within two days and can detect viable but not culturable (VNC) organisms. Most wine spoilage organisms can survive in a VNC state under the combined stress of SO2, pH, and alcohol, he said, and as the SO2 level drops in the tank or bottle, these organisms can resume growth and cause the wine to spoil.
Some wineries staff their labs with trained enologists, Burns said, but they often find that the enologists’ time is more valuable in the wine cellar making winemaking decisions. Others staff their lab with technicians to take care of basic routine analyses that are needed with rapid turnaround (less than 1 hr) and outsource larger volumes for routine monitoring to companies such as ETS.
ETS was the first wine laboratory to earn ISO 17025 certification, meeting specific international quality standards, Burns said. At peak of harvest, the company runs 1,500 samples, with 5–6 analyses each, in a 12-hr day. This is not an extraordinary throughput for a clinical lab, he said, but it is for the food industry. The company has an active research project to enhance those analyses and tie them to real-world sensory impact, he said. It is spending more than 20% of its gross income on R&D to continue improving enology methodologies and adding new ones. Burns said they are particularly interested in developing methods for newly discovered flavor and aroma compounds significant to the wine industry for which robust analytical methods don’t exist.
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Examples of Wine Analytical Systems
Many companies offer instruments for chemical analysis. Here are a few of the systems offered specifically for wine analysis.
• Anton Paar (www.anton-paar.com) offers a wine analysis system called Alcolyzer Wine that determines the alcohol content of a wine sample quickly and easily. Its NIR measuring method is said to eliminate the influence of other sample constituents on the alcohol measurement. When combined with a density meter, it determines additional parameters in a single cycle.
• Foss North America (www.foss.us) offers several systems for wine analysis. The WineScan™ FT 120 uses flow injection analysis and FTIR to deliver results in 30 sec for all major quality parameters in a single analysis, including ethanol, total acidity, volatile acidity, malic acid, pH, lactic acid, glucose, fructose, and reducing sugar. With an autosampler, the system provides unattended analysis of up to 120 samples/hr. The FIAstar™ 5000 modular system uses flow injection analysis and FTIR to provide for the simultaneous determination of up to three parameters.
• Hanna Instruments (www.hannainst.com) offers automatic titrators that can perform 25 different tests on wine, including SO2, total acidity, pH, color, phenols, percent alcohol, volatile acidity, and others. Hanna’s Multiparameter Wine Analysis Titration Systems HI901 and 902 are automatic titrators that feature Chip-Lock™ exchangeable burettes that prevent cross contamination when changing reagents for different analyses.
New Developments in Wine and Grape Juice Analysis
The following organizations offer some recent developments related to wine and grape juice analyses.
• Thermo Fisher Scientific Inc. has released an application note titled “Direct Analysis of Red Wine Using Ultra-Fast Chromatography” that describes the use of the company’s Accela™ ultra-high-pressure liquid chromatograph and the LTQ Orbitrap XL™ hybrid mass spectrometer to identify and quantify antioxidant constituents in red wine. The application note is available at www.thermo.com/redwine.
• Ohio State University researchers have demonstrated the feasibility of using Fourier Transform Infrared (FTIR) spectroscopy and chemometrics software to rapidly determine the percentage of Concord grape juice in commercial 100% grape juice blends. Christian Sweeney, Monica Giusti, and Luis Rodriguez-Saona reported their results in paper 057-14 at the 2009 IFT Annual Meeting.
• University of Missouri researchers have developed a rapid method for simultaneously quantifying sugars, organic acids, and ethanol in wine, using high-performance liquid chromatography with ultraviolet and refractive index detection. Lakdas Fernando and Ingolf Gruen presented their results in paper 057-20 at the 2009 IFT Annual Meeting.
• University of Guelph researchers investigated the effects of temperature, storage time, and packaging type on the quality of bag-in-box white wine, using oxygen transmission rate and FTIR to analyze color, free and total SO2, total aldehyde, and total phenol. They analyzed the spectra chemometrically and concluded that the resulting models can potentially be used as an efficient tool to evaluate the quality of wine. Y. Fu, L.-T. Lim, and P.D. McNicholas reported their results in the September 2009 issue of Journal of Food Science (Vol.74, pp. C608-C618).
• University of California, Davis, has begun construction of its new Research and Teaching Winery and the August A. Busch III Brewing and Food Science Laboratory. The new facilities are part of the UC Davis Robert Mondavi Institute for Wine and Food Science, and will be used for scientific research, student training, and industry collaboration. The three academic buildings of the institute, which house the Dept. of Viticulture and Enology and Dept. of Food Science and Technology, opened in fall 2008. The 34,000-sq-ft building housing the winery and the laboratory will be completed in 2010. The winery will include a large experimental fermentation area, controlled temperature rooms for large-scale testing, barrel and bottle cellars, a testing lab, a classroom, and a special bottle cellar for donated wines. The winery will be used for research, teaching, and industry short courses.
• North Carolina State University will present a Juice and Wine Analysis short course on May 13–14, 2010. Designed for beginners, the course will provide hands-on experience conducting and interpreting analyses that should be performed in winery laboratories from harvest through bottling. More information is available by contacting [email protected] or [email protected].
by Neil H. Mermelstein,
a Fellow of IFT, is Editor Emeritus of Food Technology