Don't be Fooled - Malic Acid Testing

I recently attended the Unified Wine & Grape Symposium in Sacramento.  A few winemakers I spoke with asked about the precision of the Accuvin Malic Acid test kit.  As has been reported, the accuracy of the Malic Acid test kit is +/- 10 mg/L at the 30 mg/L level, which is generally considered the end of MLF, and about +/- 20 mg/L at the 75 mg/L level.

These questions reminded me of the of the Wine Industry Interlaboratory Testing Program that has be going on since 1999.^1,2,3  The program, set up by the ASEV and UC Davis, is meant to help winery labs improve the quality of their results.  In this program, two wine samples are sent out a few times a year to the 40 – 55 participating winery labs.  The samples are analyzed at least in duplicate, and the results are sent back for statistical analysis.  The most common method used by far was enzymatic.  A table of the results is given below for wine samples with malic acid levels below 500 mg/L.  From 10% to 23% of the results were found to be unreliably inaccurate, and are not included in the table.

What do these results show?

First, in the nine years the quality improvement program has been going on, the results have gotten progressively worse.  The coefficient of variation, a measure of lab to lab reproducibility, has doubled.  In addition, while in the initial years 10% of the reported results were so far off the mark that they had to be excluded, the number of totally unusable results climbed to 23% in the latest report.

Second, the results have deteriorated to the point where about half are flat out wrong regarding what they tell the winemaker.  In 1999, a sample with a malic acid level of 100 mg/L was analyzed, with the results showing a range of 63 – 137 mg/L.  That’s pretty broad.  In 2007 a wine with a malic acid level of 110 mg/L was analyzed, and the results ranged from 30 – 200 mg/L.  In 2008 a sample with a malic acid level of 96 mg/L was analyzed, and the range for the results had ballooned to 18 – 178 mg/L.  Some winemakers were told their wines had finished MLF (which they hadn’t), while other winemakers thought they had a few weeks yet to go (which they didn’t).  And remember, that huge range was the range reported AFTER 23% of the results had been discarded for gross unreliability.  (By the way, if an Accuvin test strip had been used to analyze the above samples, you could expect the results to be in the range of 75 – 125 mg/L.  That’s considerably better!)

In 2002 Dr. Christian Butzke of UC Davis wrote a critique of the first few years of the program.  Regarding malic acid he wrote:  “Given the corresponding consequences of uncertainty over incomplete or deliberately prevented malolactic fermentation, in particular CO2 evolution in the bottle and changes in perceived acidity, this performance requires major improvements.”¹ What would he say today?

Don’t be fooled!  Spending money on expensive equipment or excessive time running laboratory tests does not ensure reliable results.

A final thought:  just getting the same answer twice with a method performed unreliably does not make it the correct answer.

1   C. E. Butzke, Am. J. Enol. Vitic., 53 (20: 163 – 169 ) 2002.

2   C. E. Butzke, Practical  Winery & Vineyard, Jan. Feb. 2002

3   Collaborative Testing Services, Inc., www.cts-interlab.com/wine/index.html, reports, 2003 - 2008

Recently we emailed several wineries regarding the Accuvin Quick Tests kit for Malic Acid analysis.  One response contained the following statement:  “I use Megazyme products and charge my clients accordingly.”

Wow!

We live in a very competitive world, and in very uncertain times.  I’m sure the clients referred to above are happy to pay extra for a wine because the winemaker has arbitrarily chosen to use a more expensive analysis method.

The Accuvin Malic Acid test strips for monitoring malolactic fermentation are less expensive than the historic UV enzyme kits, like Megazyme’s.  They don’t require a spectrophotometer.  They don’t require sample pretreatment.  They don’t require special laboratory training.  They only take 4 minutes from sample to result.  And since they don’t require any equipment, they can be used right beside the barrel or tank that needs testing.  Because of the low cost, each barrel can now be conveniently tested rather than using the method of mixing samples from 5 barrels, and averaging the result.  Our products fit right into your current winemaking regime.  And by the way, Accuvin’s Malic Acid uses traditional enzyme analysis.  It just offers the tests in a more convenient, ready to use, easy to use format.

Accuvin understands that some winemakers are interested in immediately adopting a new technology they feel will provide them with better control of the winemaking process, or provide a savings in time and/or cost.  Other winemakers would like to evaluate a product before adopting it.  Accuvin is happy to accommodate its customers, and does provide samples for comparative studies.

I recently received a question regarding the performance of the Accuvin Malic Acid test kit.  A customer had prepared a 75 m/L malic acid standard from a quantity of repackaged malic acid he had purchased.   He assumed the malic acid was the DL-malic acid form, which is about 50% active in enzymatic tests like the Accuvin test kit. 

He tested the sample using the Accuvin kit, and assumed the result would be about 30 mg/L (actually 37.5 mg/L).  The actual reading was 75 mg/L. 

What could have happened?  The malic acid he used had been repackaged.  It most likely was the L-malic acid form, which is 100% active in the enzymatic test, and not the DL-malic acid form he expected.

Chemically, the two forms are identical.  They differ in some physical properties. For example, the melting point of the L-malic acid form is 101C – 103C, while the melting point for the DL-malic acid form is 130C – 133C.

The key difference is biological.  DL-malic acid, a mixture of D-malic acid and L-malic acid, is the form most readily available commercially, and is the most economical. In DL-malic acid, the D-malic acid form will affect the acidity just like the L-malic acid form, but it will not later undergo malolactic fermentation. Thus, only about half can later be converted to the softer-tasting L-Lactic Acid; the other half is microbially stable. It should be added that the D-malic acid form has caused a slowing of primary fermentation by some strains of yeast. Also, especially when MLF is not planned, with malic acid additions there is a risk of developing green acid taste.

The bottom line:  plan your chemical additions.  If you want to make an acid adjust only without malolactic fermentation, DL-malic acid is a viable option.  If you want to adjust acid levels prior to primary fermentation, and/or if you are planning MLF, it might be better to stick to tartaric acid or use only the L-malic acid form.

Winegrape growers and winemakers have long sought a method for selecting the optimum time to harvest winegrapes.  Measurements of sugars (Brix, or soluble solids), pH, acidity, anthocyanins, berry weight, glutathione, arginine, protein levels, and terpenes, among others, were made, but they did not correlate very well with optimum wine quality.  Some researchers looked for combinations of factors, and found that ºBrix x pH, ºBrix x pH², and especially ºBrix/acidity were much more useful in making harvest decisions than the individual factors alone.^1-6 These factors continue to be recommended by major reference books.^7-11 When using the ºBrix/acidity ratio, values in the range of 30 – 32 have proven optimal, although best time to harvest varies slightly between varieties and between general climate.

Some additional techniques have been developed, such as gas chromatographic analysis of aroma components, spectrophotometric measurement of anthocyanin levels, and near infrared analysis of specific phenols.  For now, however, these techniques are at best research oriented, off line methods.

There is one method that has recently gained favor among some winemakers as a means of judging time to harvest:  sensory analysis of grapes, otherwise known as tasting.  A September 5, 2006, Wine Industry Symposium on this technique was recently described in “Wine Business Monthly.”¹² There are two schools of thought on this technique.  First, at an earlier conference discussing the technique of using taste to judge optimum time to harvest, Dr, Richard Smart stated "Winemakers go into the vineyards and they chew on some grapes, and they look at the seeds and the stems, and they mumble a few things, and then they proclaim the harvest date. And curiously, that date is always in the future, never in the past. I never heard a winemaker taste the grapes and say, 'Darn, we got here too late’."¹³ Also, in a recent study expert wine tasters showed considerable variation in their assessments of wine quality.¹⁴ 

What do I suggest?  Perhaps we should follow the advice of the late renowned French enology researcher Emile Peynaud:¹⁵  “Attempts have been made to express the state of maturity of the grape using certain ratios between different ingredients.  The sugar/acidity ratio is the simplest and most meaningful.  Since sugar levels rise while acidity diminishes during ripening, the sugar/acidity ratio rises sharply and develops more quickly than its two factors.  Although the accumulation of sugars and the combustion of acids not being subject to the same factors, take place independently in the grape, this index is a fairly accurate expression of the state of ripeness.  Its value depends on the variety.”  And if there has been a change in weather conditions such as the very cool spring this year in Oregon, perhaps doing a little extra canopy manipulation (e.g., leaf removal) will produce much better grapes than simply letting them hang for an extended period of time.

What do you think?

1.   C. S. Ough, V. L. Singleton, “Wine quality prediction from juice Brix/acid ratio,” Am. J. Enol. Vitic., 19 (3): 129 - 138 1968.

2.   C. S. Ough, C. J. Alley, “Effect of Thompson seedless grape maturity on wine composition and quality,” Am. J. Enol. Vitic., 21 (2): 78 - 84 1970.

3.   J. Ribereau-Gayon, E. Peynaud, P. Ribereau-Gayon, P. Sudraud, Traite d’oenologie sciences et techniques du vin, vol. 2, Dunod, Paris, 1975.

4.   B. G. Coombe, R. J. Dundon, A. S. Short, “Indices of sugar-acidity as ripeness criteria for winegrapes,” J. Sci. Food Agr., 31: 495 - 502 1980.

5.   C. S. DuPlessis, P. C. Van Rooyen, “Grape maturity and wine quality,” S. Afr. J. Enol. Vitic., 3 (2): 41 -45 1982.

6.   P. C. Van Rooyen, P. C. Ellis, C. S. Du Plessis, “Interactions between grape maturity indices and quality for Pinotage and Cabernet sauvignon wines from four localities,” S. Afr. J. Enol. Vitic., 5 (1): 29 - 34 1984.

7.   B. W. Zoecklein, K. C. Fugelsang, B. H. Gump, F. S. Nury, “Wine Analysis and Production,” Chapman and Hall, 1995.

8.   R. B. Boulton, V. L. Singleton, L. F. Bisson, R. E. Kunkee, “Principles and Practices of Winemaking,” Chapman and Hall, 1996.

9.   P. Iland, A. Ewart, J. Sitters, A. Markides, N. Bruer, “Techniques for Chemical Analysis and Quality Monitoring During Winemaking,” Patrick Iland Wine Promotions, 2000.

10.  J. L. Jacobson, “Introduction to Wine Laboratory Practices and Procedures,” Springer 2006.

11.  P. Ribereau-Gayon, D. Dubourdieu, B. Donneche, A. Lonvaud, “Handbook of Enology,” Vol. 1, 2006

12.  M. Greenspan, “Assessing Ripeness through Sensory Evaluation,” Wine Business Monthly, Nov. 2006.

13.  D. Berger, “”Water into Wine: the smoking gun,” Wines & Vines. 86 (3): 52 – 56 2005.

14.  R. Gawel, P. W. Godden, “Evaluation of the consistency of wine quality assessments from expert wine tasters,” Aust. J. Grape & Wine Res., 14: 1 - 8 2008.

15.  E. Peynaud, “Knowing and Making Wine,” Wiley-Interscience, 1982.

About me:

I am an analytical/clinical chemist by training, and have spent 30 years developing and manufacturing diagnostic testing products.  I switched from laboratory-based testing to point of care testing for humans, and most recently adapted the human diagnostic testing techniques to wine analysis.  I am the founder and chief of product development for Accuvin, LLC.

My current interests are wine and food analysis, winemaking, and simplifying the science of winemaking.

Thanks for checking out my blog.  I hope to offer much food for thought. 

 
Accuvin History:

 The idea for Accuvin came with the first harvest from my vineyard.  I had about a half a ton of premium Paso Robles west side Cabernet sauvignon, and I needed to do something with it.  I talked with one of the local commercial winemakers, and he offered to help me make my first wine. In return I offered to do his lab testing for him.  When shown his “laboratory” – really, a small counter in the corner of his office – I was surprised.  The collection of cylinders, burettes, beakers, and a pH meter he had were not what I expected.  In the medical testing area where I had been working, laboratory work involved either fairly expensive equipment for detail work, or simplified test devices for bedside and other point-of-care analyses.  The beaker and burette had been pushed to the side ten to twenty years earlier.

As I worked on the needed testing for the crush, I became curious and contacted other wineries to see if they had moved to more modern techniques.  Well, they hadn’t.  Simple test devices were just not available for wine analysis.  So, I thought maybe I could simplify my life by applying some of the technologies of medical testing to wine testing.

 The first step was interference removal.  Nobody wants to be bothered with some pretreatment step requiring careful measurement and timing.  In medical testing the main culprit was red-colored hemoglobin; in wine testing it was red-colored anthocyanins.  I found a way to do this in a test strip format with a technique similar to that found in the products diabetics use to self-monitor their blood sugar levels.

Following this I adapted some chemical methods to the test strip technology.  The result was a family of tests that included pH, residual sugar for monitoring the end of primary fermentation, and L-Lactic acid and Malic acid for monitoring the start and completion of malolactic fermentation.

Some analyses were not readily transferable to the test strip format.  For these a tube format was best.  For example, I formulated a mixture of color indicators and premeasured titrant to develop a one step test for titratable acidity.

Using these tests I was now able to monitor grapes for optimum time to harvest, and follow key parameters of the winemaking process, in each case getting answers in less than five minutes without cumbersome titrations with a burette and without having to purchase a spectrophotometer or send samples for expensive commercial analysis.