Malic Acid & Chromatography

box of grapes

By: Tom Payette – Winemaking Consultant 

Malic acid is one of the major acids in grapes used to make wine.  In most cases it is secondary only to tartaric acid in quantity and then followed by succinic, citric, fumaric and other small quantity acids all contributing to a total acid or titratable acidity. 

  Every variety of grapes has a potential difference in the amount of malic acid that may proportionately exist in the grapes at harvest and much of this is predicated by the growing season, amount of rainfall, overall heat summation temperatures and night time temperatures.  Cool climates often have grapes higher in malic acid and warmer climates often have lower quantities of malic acid in the fruit.  Riper fruit generally has less malic than under ripe and so on; yet, winemakers should be cautioned not to try and use a measurement of malic acid as a sole predictor to grape ripeness for winemaking.  Further note fruits other than grapes such as cherries and apples have wide ranges of malic contents.  Cherry’s and apple’s principal acid is malic.

  In some traditional roles of winemaking such as wines made from the Bordeaux varieties, Burgundy varieties and Rhone varieties a decision by the winemaker may need to be made as to whether or not to perform a malo-lactic fermentation on those wines.  In making that decision, many factors come into consideration:  How much malic is present?  What is the pH now?  What will the pH be after alcoholic fermentation?  What is the predicted pH to be after malo-lactic conversion?

Malo-lactic Fermentation

  Unlike what the term indicates, this is not a fermentation done by yeast.  This process of converting the harsher malic acid, the acid dominant in most apples, to lactic acid, the softer acid dominant in most milk and cheeses is done by malo-lactic bacteria. This small bacterium is a Leuconostoc

oenos  and predominantly responsible for the “sterile filtration” standards of 0.45 absolute microns used as a wine industry standard today.  These small bacteria, often rampant in nature, can cause serious issues to any wine in the bottle or cellar that may undesirably undergo a potentially unwanted, uncontrolled wild fermentation.

  Many winemakers today control the malo-lactic fermentation process through cleanliness, pH, sulfur dioxide, temperature and controlled conditions to support or suppress the bacterial growth. Outside of these conditions, winemakers often select a desired malo-lactic bacterium to perform the desired job, giving a wine a desirable flavor and aroma attribute, while eliminating malic acid from the wine, or less likely, the must/juice.

Supporting the Growth

  Malo-lactic bacterial fermentations can be a challenge to any cellar.  Humorously, in some cases a winemaker who wants to suppress a malo-lactic will find one starting spontaneously and one that wants to encourage a malo-lactic will find the microbe to be stubborn.  The author has seen a huge correlation toward the microbes’ growth in relationship to the pH, temperature of the wine at inoculation and the temperature during microbe growth, sulfur dioxide use and timing of encouraging the process.  The pH of wine should be in certain recommended ranges hopefully above 3.10. 

  If below this pH, the microbe may be in serious jeopardy of surviving to do its functions of converting malic acid.  The temperature of the wine for a successful Malo-lactic should be slightly above 70 degrees F if possible.  This temperature (72 degrees F) will allow the microbe to perform rapidly and with success.  T he malo-lactic bacterial fermentation should be complete in twenty days or less on the average.  The sulfur dioxide of most wines must be relatively low from near zero ppm to about 15 or 20 ppm at a maximum.  Levels above 15 or 20 ppm may show signs of no to sluggish or incomplete activity.  The timing of a malo-lcatic is often best just after alcoholic fermentation has taken place.  This often turbid, nutrient and yeast rich solute can be a healthy environment for the microbe to grow and succeed consuming malic acid.

Suppressing Malic Bacteria Growth

  Referencing the above paragraph, many readers can draw their own conclusions on how to suppress the malo-lactic fermentation.  Colder temperatures especially below 50 degrees F will help slow or stop the bacteria, a free sulfur dioxide of 35 parts per million (PPM) or higher may help suppress the microbe and lower pH’s offer a more hostile environment to the microbe.  Most winemakers use temperature and sulfur dioxide adjustments to suppress a spontaneous malo-lactic before resorting toward potentially undesired pH adjustments in a must or wine.

PH Shifts During Malo-lactic

  The shift of a wine’s pH after a successful malo-lactic fermentation is difficult to predict.  Many times a wine pH may go up by 0.1 or even as high as 0.20 pH units or more.  This is dependent on the amount of malic acid content in the wine and how much was converted during the malo-lactic fermentation.  One must recognize at this time also that there are two different configurations of malic acid.  One is consumable by the malo-lactic microbe and one is not.  Therefore, some winemakers that have performed what they believe to be a successful complete malo-lactic fermentation process may, after performing a quantifiable test on the malic acid content, find some malic acid is indeed left of over.  This can be in the range of 22-30 milligrams per liter.  Each winemaker is left to his or her own decision as to what is an acceptable level and risk.

Filtration

  Winemakers that know their wines have come into contact with the malo-lactic bacteria often elect to filter those wines to a pore size of 0.45 microns.  We often show these wines as ML positive on our cellar inventories.  Other winemakers often assume, wisely so, that any wine in their wine cellar with malic acid is considered ML positive to be on the safe side.  With these assumptions, all the wines that have any malic acid in them should be filtered to a 0.45 micron level – just to be certain.

Paper Chromatography

  A non-quantifiable inexpensive process to measure malic content in wine, must and juices is the use of paper chromatography.  Winemakers are urged to use this crude, easy process in their cellars to measure their progress of a malo-lactic fermentation.  After using this “in house” testing technique winemakers are best served to measure the amount of malic acid in their wines in some quantifiable way to get an exact number to understand how much malic is still present in a wine or not.  Well-funded internal winery labs may have the tools in their own lab to measure this acid.  Others may find it cost effective to ship a small sample to an outside lab and have it measured at an external laboratory. 

Process

  The process of paper chromatography is very simple and very affordable.  Acids are carried, by way of a solvent, up the paper a distance related to each acid and or it’s standard.  After the paper dries one may look at the “developed picture” to understand what wine may contain what types of acid.  This is non-quantifiable as previously mentioned.

Tools and Chemicals

•    Chromatography paper

•    Chromatography solvent solution

•    Hematicrit tubes  0.05 (AKA Capillary tubes)

•    Malic acid standard

•    Lactic acid standard (Note I am leaving

      tartaric out)

•    Sharp #2 pencil

•    Straightedge or ruler

•    Paper towels

•    Well ventilated work area

•    Wines to be tested ;   10 milliliters or more.

•    Standard lab safety gear

  Some winemaking supply stores have affordable pre-assembled chromatography kits with instructions.

Procedure

1.   Clear and clean a space in the laboratory to be your workspace.

2.   Make sure the area is well ventilated since the solvent for the paper chromatography is very pungent and possibly harmful over a large period of time.

3.   Wash your hands thoroughly, twice, before handling the paper used with the paper chromatography.  This will remove any grease or dirt that may interfere with the results and action of the chromatography process.

4.   Place several pieces of clean dry paper towel on the countertop area to be the workspace.  This will be another layer of protection.

5.   Retrieve a piece of the chromatography paper from its container being very careful to handle it by the edges only.

6.   Place the chromatography paper on the paper towel work area.  Place the days date at the paper at the top of the paper.

7.   Using the straight edge and pencil, draw a straight line about one inch above the bottom of the chromatography paper.

8.   With the pencil, make small dots about 1.25 – 1.50 inches apart across the page along the freshly drawn straight line at the bottom of the paper.

9.   Under each dot make a code to reference the intended product or standard so one will be able to know what was placed at each dot.  Eg:  M= Malic   L= Lactic   Mer= Merlot   PN= Pinot noir and so on.

10. Retrieve from the cellar representative samples of each wine desired to be tested.  Only small quantities are needed.  Less than 40 milliliters.

11. Line each standard and wine sample up in front of the chromatography paper work space from left to right to equate what was drawn on the paper.

12. Take a hematicrit tube for each wine out of the hematicrit tube storage container for these.  Handle them only by the end that will not be in contact with the chromatography paper.

13. Holding the hematicret tube by the top end, quickly dip the receiving end into the acid standard or wine sample desired to be tested for each mark on the chromatography paper.

14. Once a small amount of the acid standard or wine is inside the hematicrit tube quickly touch the pencil spot made that is labeled for that standard or wine.  Be very careful to keep this organized and that each spot is exactly what it is labeled.  If in doubt – re-sample and start over.

15. Rotate left to right until all of the spots have their corresponding liquid on them.  (Do your best to make these spots very small with hopes they do not grow any larger than the size of a pencil eraser head.  Just a quick touch to the paper is plenty.

16. Wait about 4 minutes for each spot to dry and the reapply with the same appropriate hematicrit at each spot a second small spot of resultant correspondent liquid.

17. Allow these spots try roughly 10-15 minutes.

18. Prepare the solvent holding vessel with about one half to five eighths an inch of chromatography solvent.  (This solvent may remain in this vessel for subsequent uses and it should remain fresh for about 8 months)

19. Gently lower the chromatography paper with the wine and standard sample on it into the solvent.  Make sure the paper is level so the solvent will travel equally, and at the same speed, up the paper.  This takes about three to five hours.

20. Replace the lid on the container and set the apparatus where it will not be disturbed, moved or knocked accidentally.  One may check this container from time to time in order to estimate when to remove the paper.

21. After the solvent has visibly moved 95% of the way up the paper, one may remove the paper from the chromatography solvent vessel.

22. Replace the lid to the chromatography vessel.

23. Hang the freshly solvented paper in a well ventilated area to dry.  Make sure the drying process takes place away from any sulfur dioxide, bleach or other similar chemicals.  This paper should dry 10 hours or more and most winemakers allow it to dry overnight.

24. Read the chart the next day by looking at it.  Resist the temptation to look at it up close.  The author prefers to look at it about 5-7 feet away to get a true picture of what is happening.  The results will not be well defined and often ghostly or blurred.  This is normal.

25. Relate the distance the standards traveled to the distance the acids in the wine spots travel.  Try to ignore any colors from the wine such as potential red stains, etc.

26. Remember while reading this “film” that this is only used to get an idea of whether some malic acid is left in certain wines or not.  Some wines will have a more defined spot and others may be less easy to read. 

Calculation

  There is no real mathematical calculation as one can see.  One should relate where the malic and lactic acid standard spots are on the chromatography after the drying time and look horizontal to see if resulting spots exist in the same horizontal area above the wine spots.  This is an indication there is some of the same acid in the wine.

Mixing and Standardizing Chemicals

  Due to the difficulty of making these chemicals and the reasonable cost of the chemicals, it is recommended to order the chemicals from an outside lab.  Making the solvent is especially cumbersome with separation flasks and other tedious time consuming issues.

Other Helpful Tips

  Plastic gloves and goggles may be worthwhile to use if one finds they cannot handle the paper without getting skin oils and other contaminants on the chromatography paper, etc.

  NOTE: Always perform this test in a well ventilated area – the solvent is odorous and unpleasant.

References

Amerine, M.A., Berg, H.W., Cruess,W.V. 1972. The Technology of Wine Making

Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., and Nury, F.S. 1999. Wine Analysis and Production

Verbal discussion with:  Mr. Jacques Boissenot, Mr. Chris Johnson, Mr. Joachim Hollerith and Mr. Jacques Recht.

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