Monitoring Malolactic

Bob Peak, prolific writer on fermentation science and partner at The Beverage People in Santa Rosa, Calif., runs a sample test through the shop's Reflectoquant.

Days are getting shorter. Birds dart and weave across harvested vineyards looking for stray berries to eat. Sore backs are beginning to hurt slightly less. Harvest cuts and calluses are starting to heal and slough off work-stained hands. The constant fatigue is beginning to lessen.

Shortly after the adrenaline rush of crush, winemakers across the country begin to focus on monitoring malolactic fermentation (MLF). Whether seeking assurance that red wines make it through complete MLF or white wines go through partial or no MLF, winemakers will be all over the monitoring process like Oenococcus oeni on l-malate.

Here we’ll explore how winemakers strive to reduce risk by enlisting sensible, efficient and cost-effective ways to monitor MLF, be it good old-fashioned paper chromatography, sending out samples for lab work or investing in accurate and affordable diagnostic equipment. BASIC TESTS Paper chromatography Many small artisan wineries find that paper chromatography is the answer to their MLF-monitoring needs. It yields qualitative results, meaning it shows the presence or absence of tartaric, malic and lactic acid. However, it is not quantitative, meaning it will not show precisely how much acid is in each sample.

Paper chromatography has sensitivity threshold of about 100mg/L in indicating a qualitative result, so it has its limits in determining final MLF. This method can also be used to estimate partial MLF when coupled with tasting to confirm testing results.

Depending on the grade of paper, results can take 6 to 12 hours to develop. When doing paper chromatography, testing must be conducted in well-ventilated conditions, as the process is very smelly, even as the solvent soaked paper dries.

Chromatography solution is comprised of three ingredients: bromocresol green, formic acid and butanol. It has an expiration date of one year after opening and should be stored in its original container, away from extreme temperatures. If left in a spacious chromatography jar after use, the shelf life decreases. When returned to its original container with less headspace, the shelf life increases.

When the solvent is too old, the contrast between the yellow and green areas becomes weak, or it does not develop at all. When it comes to disposal, the solvent should always be kept out of sewer and septic systems and disposed of according to state, local and federal guidelines.

Since there are different grades of chromatography paper on the market, it’s important to know the grade you buy, as developing times can vary:

Whatman Grade 1, considered the world standard in chromatography paper, has good resolution for general analytical separations. It can be left in solvent between 6 and 8 hours.

Whatman Grade 20, at almost double the price of Grade 1, is a supreme grade for maximum resolution with the greatest possible separation. It can be left in solvent for 12 or more hours.

Due to the sensitivity of chromatography paper, always store it in an airtight container, away from CO2, SO2, acid and alkaline fumes—as well as moisture and bright light.

Hoot Smith, owner of Kastania Vineyards in Petaluma, Calif., with a case run just shy of 1,000 cases per year, uses paper chromatography to monitor MLF as a first measure. When he feels it’s complete, he sends samples off to Vinquiry in Windsor, Calif., for confirmation. Even though he usually sterile filters his wine, he likes to be on the safe side.

Smith inoculates with an ML culture when the wine is totally dry in the barrel, and begins monitoring in 1.5-3 weeks to get an idea of what’s going on. By listening to the telltale Rice Krispie-like pops and crackles, he can tell MLF is under way. If the weather is cold, and things are progressing at a sluggish pace, Smith hops on his forklift and takes the barrels outside for a little sunshine and gentle fermentation-friendly warmth. It’s a commonly employed old-school version of flipping the switch on a heating jacket around a tank.

White wine vintners who don’t want MLF must start their testing immediately after alcoholic fermentation as well as consistently monitor and maintain MLF-inhibiting SO2 levels. Since gross lees can harbor nutrients and bacteria, most winemakers will want to rack right after alcoholic fermentation and keep the wine nice and cool and pH low.

Kerry Damskey, a Sonoma County-based consultant winemaker with more than 25 years of winemaking experience, oversees production for 17 wineries and produces 1,000 cases of mountain wine per year at his winery, Palmeri Wines in Geyserville, Calif. Damskey tracks malates by ordering enzymatic assays at either Vinquiry or ETS labs. His advice to smaller wineries is to use paper chromatography with a lab follow-up—the very same approach Hoot Smith uses.

Though many winemakers have great success exclusively using paper chromatography, the reality is that with a sensitivity threshold of 100mg/L, there is a chance there could be traces of malic acid still present in the wine. Especially considering the target for complete MLF can be 30mg/L with most winemakers targeting 15mg/L or less.

Dave Breeden, winemaker at Sheldrake Point in the Finger Lakes region of New York state, has been making wine for 7 years now. With an annual production of 6,000 cases (60% white and 40% red), Breeden uses paper chromatography exclusively. If MLF is ever sluggish, he sends samples off to Cornell’s wine lab for analysis. Since he sterile filters most of his wines, the qualitative results and sensitivity threshold of paper chromatography are inconsequential. In the rare event he chooses not to sterile filter, he sends samples off to Vinquiry in California for a complete lab panel, because at that point, paper chromatography would be too risky to go by exclusively.

When considering the cost of a professional lab test as a back up, and if sterile filtering is not in the plans, consider the cost of time, grapes, bottles, corks, foils, labels and labor in the event that bottled wine needs to be dumped. If the wine is still palatable, tack on time and energy explaining to customers in the tasting room why there are tiny bubbles and dusty sediment in the wine, and expect a major decrease in its sales.

Accuvin quick tests for malic acid Accuvin in Napa, Calif., offers colorimetric wine analysis tests for malic acid for $2.97 per test for 20 test kits, $2.25 per 50, and $2.08 per 100. The tests can monitor the progress as well as the completion of MLF. Accuvin’s Quick Test for malic acid enables a user to test for MLF at 75 mg/L, 50 mg/L, 30 mg/L or 0 mg/L. Results take four minutes to develop.

Accuvin Quick Tests, which debuted on the market near the end of 2002, are like a home pregnancy test or glucose test for diabetics. Instead of health indicators, they give winemakers basic information to monitor the winemaking process. The tests can be run on both red and white wines with no pretreatments such as the dilutions or color stripping required for tests on a spectrophotometer. The results are fairly quantitative, but since it’s a visual test, you have to be able to interpret color as well as consider the color temperature of the light in which you’re reading the test.

Some winemakers doubt the accuracy of Accuvin’s Quick Test, while other professionals use them as a first step in the monitoring process. Dave Crippen, head winemaker at 100,000-case Renwood Winery in Plymouth, Calif., started making wine 25 years ago.

Crippen said, “Back in the day we used paper chromatography, but it was tedious, and no one in the winery enjoyed the smell of solvent.” As a first step in monitoring MLF, Crippen is a fan of Accuvin Quick tests because they’re inexpensive and easy to use and interpret. He also said they’re fairly accurate when compared with the results of his in-house analysis on the winery spectrophotometer he uses to confirm the Accuvin results.

Justin Tatum, assistant winemaker at Camarillo Custom Crush Winery in Camarillo, Calif., oversees numerous wines at one time, 150 of which could be actively undergoing fermentation.

Due to the sheer volume of wines Tatum works with, coupled with his experience as a winemaker, he is comfortable using Accuvin Quick Tests as a first measure. In the event something doesn’t seem right, his second measure is paper chromatography, and third is sending samples off to Vinquiry for lab analysis.

Spinning a mistake

There is a white varietal winemaker who, because of budgetary constraints, used only paper chromatography as the means of analysis. Early on in the process, as well as right before bottling, this highly skilled and extremely knowledgeable person diligently tested a two-barrel batch of Sauvignon Blanc using paper chromatography. Sulfite levels were also consistently monitored. The wine had no traceable amounts of residual sugar. Being a purist, the winemaker did not intend to sterile filter this release.

Come bottling time, like a good pilot during a pre-flight check, the winemaker made certain the pH, free SO2 and alcohol levels were appropriate enough to inhibit MLF. Since test results continuously indicated an absence of malic acid, the wine was bottled in the winter. Six months later, when the weather got a little warmer, tiny bubbles starting forming and the Sauvignon Blanc began to throw a light sediment. To great dread and surprise, the remainder of MLF was going on inside the bottle. Though the wine was perfectly palatable, the winery had to explain to customers at the tasting bar that the wine was slightly pétillant, putting an elegant spin on an unfortunate mistake. This resulted in a major sales slowdown of one of only two white wines the winery offered, thus cutting into profits at a time when white wine was most sought after. K.K.

LAB EQUIPMENT Reflectoquant System EMD, known as Merck in North America, makes the Reflectoquant RQflex 10 reflectometer, retailing around $775, and 50 RQflex Test Strips for malic acid for $120. With different test strips, this system also analyzes TA, total sugar and free SO2.

Results are only useful if the wine is toward the end of MLF, as they range from 1 to 60ppm. Any result above 60ppm will register simply as “high,” which is not helpful if you wish to have your wine undergo partial MLF, say in a white wine, where a richer mouthfeel without a loss of fruitiness or acidity is desired. Most partial MLF results would read in the 500-1,000 mg/L range, however, taste will be more of a target rather than a specific number.

Due to the possibility of variation in the lots of strips and reagents, the system requires periodic calibration with each new set of strips. This is done by inserting a barcode, which comes with each set of strips, into the machine.

Bob Peak, prolific writer on fermentation science and partner at The Beverage People in Santa Rosa, Calif., for the past 7 years, ran a sample test through the shop’s Reflectoquant with me. He pointed out that in order to test a red wine for MLF you must color strip the wine first. Activated charcoal is normally used for this process, but it’s messy and requires filtration. So Peak came up with an easier option using Polyclar PVPP, an inert polymer-fining agent made of powdered nylon that’s used to correct pinking or browning of white wines. He placed a 1/2 teaspoon of PVPP in a 15ml wine sample, shook well to mix, and waited 10 minutes for the sample to settle and clarify. Next, he slowly tipped the sample vial at a 45° angle, where the liquid was about to spill out, exposing the most surface area in the decolorized layer of clarified wine. Then he dipped the two test pads on the end of the strip into the decolorized section of the sample. He inserted the strip into the meter, and a result appeared on the digital readout about 5 minutes later.

Before purchasing a Reflectoquant, make certain that the test strip kits you need are still available. Due to the economic recession, EMD was forced to discontinue many test strip kits. To reactivate a product, EMD requires a commitment of purchasing 100 kits, something that may be impossible for smaller winery budgets and struggling suppliers. At the time of this article, malic acid, TA, pH, sugar and free SO2 were still available. Also keep in mind that these 50-strip test kits have an expiration date of 18 months from the date of manufacture.

MegaQuant colorimeter The MegaQuant colorimeter by Megazyme costs $851 plus $20 shipping. Included with the device is a training video and data booklet along with 60 enzymatic tests for ML. Additional videos are available on megazyme.com. The MegaQuant can also measure D-glucose/D-fructose.

From watching a demo video of the MegaQuant on Megazyme’s website, it’s clear that using it is a precision intensive multi-step process that requires more lab equipment and skills. As noted on page 4 of the MegaQuant L-Malic Acid Assay Procedure Booklet, “The accuracy of the results will only be as good as the accuracy of the pipetting and dilution of the wine samples and the cleanliness of the glassware.”

Unless you have 5 minutes to sip a strong cup of grappa-laced coffee while stirring your test sample to dissolve a PVPP tablet, a test tube stirrer is highly recommended to accelerate the dissolution of the tablet into the sample.

The upside to the MegaQuant system is that it’s fairly accurate, and the test reagents are stable for 2 years.

EnoLyzer Unitech Scientific’s EnoLyzer is a semi-automated photometer, which debuted at the Unified Wine & Grape Symposium in January 2010 and became commercially available in May 2010 with an attractive price tag of $2,700.

EnoLyzer comes preprogrammed with nine sample protocols and can test a full menu of wine tests including: malic acid, glucose/fructose, acetic acid and total phenols using traditional enzymatic reagents. It can read, calculate and print results in 3 seconds, and it runs standard wavelengths: 340, 420, 520, 580, 620, and 750nm with alternate filters available to expand the range.

To operate this device you will need lab skills as well as a good pipettor and some basic supplies including enzyme kits, pipette tips and cuvettes.

The pros of the EnoLyzer are its compact footprint, easy-to-use touchscreen interface and built-in thermal dot matrix printer. It calculates results automatically in your preferred units of measure based on four calibration options. Other than replacing the printer paper as needed, as well as the bulb in 5 or 6 years, and routinely calibrating the system for specific enzyme kits, the EnoLyzer is maintenance free.

Geoffrey Anderson of Unitech Scientific, Hawaiian Gardens, Calif., also mentioned the importance of accurate pipetting technique and suggests that those who are new to pipetting should test the same sample a few times until repeatable results are achieved. Search the term “auto pipette” on YouTube to learn some finer points on proper techniques.

Spectrophotometer For higher volume wineries, a spectrophotometer is another way to monitor MLF, and of the above-mentioned methods, is the most accurate if done properly. In addition to the unit itself, you also will need to invest in lab supplies such as enzyme kits, cuvettes, micropipettes and pipette tips—all of which can easily cost $1,800 or more. These additional costs, some of which will be recurring, should be taken into consideration when budgeting for a spectrophotometer.

You will also need to know precisely how to use the device as well as regularly calibrate it and maintain meticulous lab protocol. Otherwise, results will be about as good as what you’d get from a Ouija Board.

The cost of an entry-level spectrophotometer is in the $2,700-$3,400 range. You can also find new, used and refurbished spectrophotometers at labx.com. Since labx.com is a market-specific listing service, it’s considered a safer place than eBay to search for lab equipment deals.

To make life a little easier, Vintessential Laboratories in Australia makes reagent kits developed specifically for the wine industry, complete with instructions. The kit for malic acid is $93 for 30 tests or $260 for 100 tests.

Reputable suppliers can help select equipment and design a lab based on a winery’s budget and needs. It is important to make sure your supplier will answer any questions you may have after receiving your instrument. Extra points if they help troubleshoot, as some suppliers will.

Lisa Van de Water, founder of The Wine Lab in Napa, Calif., currently runs Vinotec Napa, and is internationally known as “The Bad Wine Lady” for her highly specialized knowledge of microbial wine spoilage. Van de Water also developed yeast and ML nutrients, which Beverage Supply Group in Napa, Calif., is exclusively licensed to manufacture and distribute within the United States.

“It all comes down to cost-benefit analysis,” Van de Water said of whether or not to invest in lab equipment. “The number of tests you do in a year, how much time it takes, how much the instrument, materials and maintenance costs, and amortize it over a 3- to 5-year period.”

Her reasoning for using a 3- to 5-year period is, “It’s not like I’m going to leave my equipment to my grandchildren. I need to know how it will benefit my business now.”

Van de Water also had some helpful points to consider when thinking about purchasing a spectrophotometer: Make sure the spectrophotometer you select can reliably and accurately perform at 340nm, which is at the edge of the UV spectrum and is the wavelength used for analyzing malic acid, acetic acid and residual sugar. Therefore, selecting a spectrophotometer that is UV-visible is absolutely essential.

As far as optical configuration goes: single beam is good, but double beam is better. Though single and dual (also known as split) beam may be more economical, the accuracy of double beam is greater.

“Your result is no more accurate than your pipettor,” Van de Water said. Next to the spectrophotometer itself, the pipettor is key to yielding accurate results. It is also critical to get your pipettor professionally calibrated every year. Your supplier should be able to arrange service visits for a small fee.

When selecting cuvettes, the clear square tubes that hold samples, look for methachrylate instead of polystyrene. The reason being, at 340nm, polystyrene absorbs most of the light, which is a serious factor considering the analysis you’re doing is light-based. Methachrylate cuvettes look exactly like the polystyrene ones, only they’re slightly more expensive and will improve the accuracy of any enzyme test done at 340nm.

PROFESSIONAL LAB SERVICES Eastern Wine Labs Eastern Wine Labs in Southampton, N.J., offers l-malic testing for $18 per sample with a 24-hour turnaround time. They service both large and small wineries east of the Mississippi and are familiar with varieties grown in the eastern region.

Nicolaas Opdam, owner of Eastern Wine Labs, has been a professional enologist for 37 years. Many of Opdam’s smaller clients don’t have the budget to buy set analysis packages, so he offers a comprehensive analysis menu that can be customized to meet a client’s needs. After many years in the business, Opdam found that this was the most efficient and economical way to serve artisan wineries in his area.

Consulting services as a “virtual winemaker” also are available and can be billed by the day or in affordable 15-minute intervals.

ETS Laboratories ETS Laboratories in St. Helena, Calif., with satellite locations in Healdsburg, Calif., McMinnville, Ore., and Walla Walla, Wash., offers MLF testing services using their proprietary Sequential Analyzer starting at $22 for a single sample. Testing prices decrease with larger quantity test orders. Turnaround time is 24 hours.

Patricia Howe is the founder of Ascent Laboratory in Napa, which was sold to Vinquiry in 2001. She has 30 years’ experience in the wine industry and recently joined ETS Laboratories in Roseburg, Ore., where she’ll be heading their newest satellite laboratory.

Howe said, “Each winery will have specific needs for accuracy, precision, limits of detection and cost. Quite frankly, in many cases it is more economical to send some tests out to service labs rather than invest in a technology that might be beyond the means and abilities of a small winery.”

Gusmer Enterprises Inc. Gusmer Enterprises Inc., in Napa, Calif., has a minimum analysis charge of $24, with discounts for 10 or more samples of the same test. An enzymatic assay for l-malic is $18 (or $16 each for 5 or more samples). Turnaround time is usually same-day.

Slightly more in-depth analysis includes: pH, malic acid and microscopic exam for $40. Turnaround time is 24-48 hours. A problematic MLF panel includes: alcohol, VA, pH, free and total SO2, malic acid and a detailed microscopic exam for $99. Turnaround time is 24-48 hours. All samples must be sent to Gusmer’s Napa facility.

The annual Gusmer Enterprises Wine Products Catalogue, which can be downloaded from the Gusmer Enterprises website, has many practical winemaking tips as well as a detailed and informative section on MLF management.

Gusmer is also the exclusive North American distributor of the OenoFoss, a sleek little device that can analyze glucose/fructose, pH, TA, malic acid, VA and ethanol. Gusmer declined to quote an exact price for the device, but another source confirmed that it is more than $30,000. However, savings in the long run would come from the fact that you need very minimal lab gear and don’t need an on-staff lab technician, reagents or lab skills to operate it.

Scott Laboratories Scott Laboratories in Petaluma, Calif., with satellite locations in Healdsburg, Calif., and Pickering, Ontario, Canada, offers enzymatic assays for l-malic at $22 per test. They can give you accurate results at any point in the MLF process with a 24-hour turnaround time. In the frustrating event that your MLF gets stuck, they can also perform a quick malic assay, where a lab tech inoculates a sample with malolactic bacteria and checks the degradation of malic acid at 24- and 48-hour intervals. If there is 20%-60% degradation, Scott Laboratories can advise what type of ML bacteria to use to restart fermentation. An assessment of the original sample for alcohol, VA, total SO2, glucose/fructose, pH, malic and Microscan is done at the same time. The cost for this level of testing is $158.

Included in the useful Scott Lab Fermentation Handbook is a section about selecting the proper Oenococcus oeni strain for different wine types and fermentation scenarios, as well as guidelines for MLF management. The handbook, which can be downloaded from the Scott Lab website, is a handy tool when your hair is on fire during crush and you need a quick and easy-to-use guide to managing gnarly fermentation issues. When armed with good information, winemakers can learn to stop worrying and love Oenococcus oeni.

Maggie McBride, laboratory manager at Scott Laboratories, suggests that the best way for small wineries to inexpensively monitor MLF is through old-fashioned paper chromatography, then ordering an assay for l-malic for more precise results.

Vinquiry Vinquiry, in Windsor, Calif., with satellite locations in Napa, Paso Robles, and Santa Maria, Calif., offers malic testing for $16 per test. They also perform a malolactic assessment panel for stuck fermentations. This assessment, which has a turn around time of 24 hours, includes: alcohol, VA, total SO2, glucose/fructose, pH, malic and Microscan for $100.