Four methods of assessing completion of malolactic fermentation

 

This article addresses determination of completion of malolactic fermentation (MLF) for amateur winemakers and was compiled from information provided at a Saanich Sommeliers workshop in November of 2018. The primary presenter was Dr. Sandy Kirk but others present contributed.  Apart from the workshop another source of information is cited below.

Introduction

Malolactic fermentation is desirable in red wines or chardonnays for long-term stability of the wine and for enhanced wine complexity.  MLF uses the beneficial bacteria Oenococcus oeni  to convert malic acid into lactic acid.  Free SO2 in wine can inhibit the ability of this bacteria to carry out malolactic fermentation.  Therefore SO2 levels must be kept low during malolactic fermentation carrying the risk that wines may be left unprotected against oxidation and microbial contamination. As soon as the MLF is done then SO2 should be raised it to appropriate levels protection of the wine.  Therefore it is important to know then when MLF is done.

For the professional there are analytic procedures for MLF completion that are available using advanced analytic techniques and equipment.  For the amateur winemaker, however, the cost of the equipment are prohibitive and other less sophisticated methods must be employed.  Four such methods are described in this article and a fifth not discussed at the workshop is cited in a website link below.

Method 1

The first method is the least sophisticated but still utilized by many amateur winemakers.  When wine with a pH of greater than 3.3 under fermentation lock is inoculated with a beneficial Oenococcus oeni  at temperatures between 18 and 22 C  it ferments slowly producing very small CO2   bubbles.  If the amateur winemaker is using glass carboys for winemaking then these bubbles may be observed rising to the top of the carboy while MLF is taking place.  Over time  the production of carbon dioxide and gas bubbles  stops.  At this point some wine makers, believing that MLF is complete, will add metabisulfite to achieve protective SO2 levels. Obviously this method is limited to fermentation in glass carboys where CO2 bubbles can be observed and it lacks analytical evidence of successful malolactic fermentation.  Additionally,  in some instances the source of the bubbles could be other than MLF. 

Method 2

To produce analytical evidence an additional step that can be undertaken at this point using the same equipment and titration solutions that were employed in initial measurements of juice or must prior to alcoholic fermentation.   Malolactic fermentation causes a slow reduction in titratable acidity.   Malic acid is a diprotic, lactic acid is monoprotic.  Assuming 50% presence of malic acid in a total assets in a wine (see Basic Wine Chemistry article on this website), then if the initial titratable acidity is 8 at the onset of fermentation it will drop by 25% to about 6 on completion of MLF.  

While providing evidence of MLF occurrence,  the above described measurements have a high  potential for error as the initial measurements are taken before alcoholic and malolactic fermentation  commenced and  secondary measurements are taken some time later at the end of MLF.   In addition, when conducting the analysis dissolved CO2 can contribute to erroneous results.   A resourceful winemaker may, however, find ways to reduce error in  measuring titratable acidity and make this method useful.

Method 3

An improvement on the ability  to determine whether or not malolactic fermentation is complete is paper chromatography. Paper chromatography kits are available online and can be often ordered from a local supplier.  The principle of chromatography is migration of chemicals of different molecular weights by a  solvent on a paper medium.  In this case the solvent is specific and must be stored in tightly closed container on 10% formic acid and decanted from it prior to use to use.  The  illustration  demonstrates how the standards and test samples may be laid out on the paper. 

Here are some tips:   

  • When known standards and wine samples are placed on the chromatography paper they should be about 2 mm in diameter.  One could use a toothpick to produce that size of sample or a micro-hematocrit capillary tube which should be available from medical suppliers.  
  • The ends of the paper should then be stapled together to form a cylinder.  
  • Pre-saturate the jar in which the chromatography will be completed and the paper with water vapour.  
  • Place the chromatography chromatography solution into the chromatography vessel making sure that only the coloured upper solution and not the formic acid gets into the vessel.  
  • Run the test until the solution reaches to the top of the paper no further. 

Remove the chromatography paper and let dry overnight in a location where solvent fumes will not be a concern, and read the next day when dry  Compare wine sample analysis with the known standards.  The location of malic acid and lactic acid will be evident in the sample being analyzed.  Where there is no indication of coloration in a test sample when compared to the malic acid standard, and there is coloration for  lactic acid when compared to the standard standard it is assumed that MLF is complete.   

When running multiple samples on one  sheet of chromatography paper this method of analysis is relatively cheap.  In addition chromatography chemicals are stable for long periods of time.  A demonstration of a paper chromatography test using several samples was provided in the workshop.   In one of the samples of a wine that was assumed to have completed MLF was shown to have residual levels of malic acid 

 

Method 4

The fourth method of assessing completion of malolactic fermentation is a quick test kit produced by Accuvin in Napa California.  This company makes a number of different quick tests for winemaking using colour developing test strips.  With the malolactic test kit the intensity of the colour is proportional to malic acid levels.  This test is simple to perform with results available after a few minutes.  The test kits are readily available and easy to store.  

A demonstration of the Accuvin malic acid test was conducted at the workshop.  There was correlation  between the Accuvin test and the paper chromatography test of the sample with an incomplete MLF,  although assessing the colour of the results when compared to the colour standards in the kit created a point of discussion by those reading the test. 

There are two disadvantages to the quick test kit.   The first is the price; each test is relatively expensive.  The second is that the test kits have a shelf life that may not permit the total consumption of a kit by an individual, further adding to the price of each test.  Both of these disadvantages can be mitigated and the Accuvin test kits are a reasonable choice to assess MLF completion in situations where strips can be used prior to expiry.  

 

There  a fifth method that was not discussed, and that was malic acid detection by the Vinmetrica method.  It is mentioned in this article because Vinmetrica claims  in the reference below that above the initial cost of purchase of equipment, the individual tests are similarly priced to the Accuvin tests and enjoy a higher degree of sensitivity.  For an interesting read on test sensitivity of wine malic acid testing please see this reference :

Vinmetrica MLF Testing Comparisons