Thursday, June 12, 2014

Monitoring the malolactic fermentation of wine

I have recently described the malolactic fermentation process. In general, a winemaker needs to be able to tell if/when MLF has commenced, how it is proceeding, if any problems are in the offing, and when it has concluded. Key to addressing these concerns are the establishment of benchmarks for the beginning and end of MLF and a regime for tracking its progress. 

According to Boulton et al. (Principles and Practices of Winemaking, Chapman and Hall, 1996), there is a difference in perception as to what constitutes the MLF duration depending on whether you are a microbiologist or a winemaker. The microbiologist sees MLF as commencing with the “introduction of viable bacteria into the wine or must” and ending “when the bacteria have gone through the growth phase and have re-entered their final resting or stationary phase.” The winemaker, on the other hand, will equate the start of fermentation with a noticeable drop in malic acid levels (grape juice contains between 1 and 8 g/l malic acid) and as completed when the malic acid has finally disappeared ( (Malolactic Fermentation Monitoring -- Resources for Winemakers) recommends a figure of < 0.05 g/l as safe for declaring the end of MLF while Katherine Mansfield (Monitoring Malolactic Fermentation 3 Ways, Cornell University College of Agriculture and Life Sciences, Cornell Cooperative Extension) cites a number of 30 mg/l).

Even though acknowledging the microbiologists' view of MLF duration, Boulton et al. accept the winemakers perspective in that they see the “measurement of the disappearance of malic acid as the accepted means for determining whether the malolactic fermentation has occurred.” Lerm et al., place this measurement of the presence of malic acid within a broader context: “The continuous monitoring of MLF is essential and often neglected by winemakers.” Continuous monitoring “allows the winemaker to follow the progress of malic acid degradation as well as the bacteria responsible for the fermentation. This is also a way for the winemaker to identify any difficulties before they can affect the quality of the wine.”

There are a number of tools that are available for the monitoring of MLF and they are summarized in the table below. 

Selected MLF Monitoring Techniques and their Attributes
Monitoring Technique
Paper Chromatography (PC)
  • separate compounds based on their polarity
  • visually follow disappearance of malic acid
  • commonly used in winery

  • easy to use
  • simple, affordable and indicative of MLF progress

  • strictly qualitative so still need quantitative values to verify MLF completion
  • not precise
  • not specific for L-malic acid
Thin Layer Chromatography (TLC)
- similar to PC but uses TLC plates instead of paper

  • easy to use
  • simple and affordable
  • results in one hour,; much faster than PC

  • not precise
  • not specific for L-malic acid
  • strictly qualitative so still need quantitative values to verify MLF completion
  • Reflectoquant®
  • based on reflectance photometry
  • use reactive test strips to analyze for various wine components

  • a fraction of the cost of a spectrophotometer
  • half the cost of an enzymatic kit
  • measure multiple wine parameters
  • fastest method currently available (5 min/sample)
  • relative accuracy of 10%

  • measure relative malic acid levels so still need to qualify absolute levels
  • operating range 1 to 60 mg/L, so some samples need to be diluted or decolorised
  • need to be calibrated with reference method
Enzymatic Analysis
  • uses enzyme that specifically react with L-malic acid then use UV-visible spectrometer to monitor enzymatic reaction
  • most commonly used method
  • MLF complete if malic acid is less than 200 to 300 mg/l

  • quantitative
  • excellent precision
  • kits readily available
  • quantify very low levels of malic acid
  • results in 30 minutes

  • more complex
  • more expensive
  • short shelf life of reagents after activation
  • require use of accurate micro-pipettes
  • turbid samples need to be centrifuged
Capillary Electrophoresis (CE)

  • highly accurate
  • short analysis time; fast results

  • extremely expensive
  • not recommended for everyday use in winery
Fourier-transform Infrared (FT-IR) Spectroscopy
- use infrared spectra to quantify wine parameters

  • accurate
  • small sample volume
  • short analysis time, fast results

  • expensive equipment
  • accuracy dependent on reference values and calibration curve
High Performance Liquid Chromatography (HPLC)
- separation of compounds based on polarity and interaction with stationary or solid phase

- highly accurate

  • extremely expensive
  • not recommended for everyday use in winery
Source: Lerm et al., Table 7.

Of the mechanisms listed above, the one that is most commonly used in wineries today is paper chromatography. Paper chromatography detects the presence of malic acid but does not tell its concentration. As such, it should never be used to make a decision regarding the end of MLF. According to Mansfield, the lower level of malic acid detection for paper chromatography is 100 mg/L, way above the 30 mg/L considered by her lab to be the “safe number” for the end of MLF. The risk associated with prematurely calling the end of MLF is residual malic acid which can be metabolized by latent LAB in the bottle with a host of resultant problems (change in color, aroma, flavor; CO₂ production; clouding (Lisa Van de Water, Monitoring microbes during cellaring/bottling,, January/February 2010)). In a table accompanying the article, the author shows that Oenococcus would be found at levels of 500+/ml if residual malic acid were present. Ms. Van de Water suggests checking wines during MLF for the presence of spoilage bacteria and doing so by microscope as well as by PCR.

A robust MLF monitoring protocol which incorporates some of the tools listed in the table above is as follows:
  1. Take representative samples of cellar barrels for testing. Care should be taken to ensure that ease of access does not determine the barrels utilized for sampling.
  2. Test consistently.  I propose that the sampling be conducted every two weeks or at topping.
  3. Position paper chromatography as a tool in the MLF monitoring toolbox for measuring progress. Paper chromatography detects the presence of malic acid but does not tell the concentration. The level of malic acid in the wine needs to be below 30 mg/L in order for the microbial stability benefits of the MLF process to accrue. 
  4. Utilize enzymatic analysis in the MLF monitoring protocol as a means of certifying the end of MLF. As the cost of the equipment for enzymatic tests are very high, I recommend the services of an outside lab for this purpose. As recommended by, the costs of these outside tests can be minimized by submitting barrel composites. If no malic acid is detected, then all of the barrels will be assumed to have completed MLF. If the test comes back positive, then the barrels would be tested individually in order to identify the offending barrel(s).
  5. Include the human factors (nose, palate, and skills; Dr Wann, Power Point presentation) in the MLF monitoring process. Dr. Wann recommends: (i) checking for CO₂ evolution; (ii) smelling and tasting the barrel at every topping (heads up on potential spoilage activity); (iii) maintaining an appropriate temperature (between 18 and 22℃); and (iv) to be aware of increasing pH levels (potential foothold for spoilage organisms). In that CO₂ is one of the outputs of the MLF process, its presence is indicative of ongoing malate degradation.
  6. Institute a number of post-MLF processes to ensure the microbial stability of the wines through to the blending and bottling processes. According to Wibowo et al., additions of SO₂ and storing the wine at higher temperatures leads to the progressive loss of viability of any bacteria surviving the MLF. The wines themselves may be sterilized by filtering with membranes having pore sizes of 0.22 to 0.45µm. Lafon-Lafourcade et al., sees the decline in LAB accelerated by increasing temperatures, lowering the wine pH, and increasing the alcohol concentration, actions which, it seems, combine to provide a toxic environment for the bacteria. The addition of SO₂ does result in a rapid loss in cell viability, they agree, but growth recommences at a later date. The specific post-MLF operations that should be undertaken, according to Boulton et al. are as follows:
    • Transfer the wines off the lees and a rough filtration
    • Adjustments of temperature and pH and addition of SO₂. The recommendation here is for 0.8 mg/L
    • Fining operations could also be performed at this time
Following this set of procedures will minimize the potential for microbes indicating their presence in the bottle.

©Wine -- Mise en abyme

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