Malolactic Fermentation Monitoring

Introduction

Accurate monitoring of Malolactic fermentation (MLF) is one of the most critical analytical parameters in red winemaking, but one that is commonly overlooked prior to bottling. We are often asked to investigate causes of red wine spoilage, and one of the first questions we ask the winemaker is “what was the malic acid concentration prior to bottling?” Unfortunately, many winemakers simply do not bother to check or assume that MLF has gone to completion based on no more visible signs of fermentation, or by using qualitative methods such as Paper Chromatography.

Types of test available

This paper will only discuss methodologies available to small to medium-large wineries along with their strengths and weaknesses. This includes;

Paper Chromatography
Thin Layer Chromatography (TLC)
Enzymatic analysis

A review of more advanced and complex methodologies such as HPLC or Segmented Flow Analysis will not be covered since these methodologies are applicable only to a few very large wineries.

The advantages and disadvantages of each test are summarised in Table 1. Five key areas are examined,

Cost (includes capital, labour and consumables)
Complexity (operator skills required, interpretation of results)
Accuracy (qualitative versus quantitative)
Speed (result turnaround time)
Reagent stability

Table 1 Comparison of Available Methodologies

	Costs	Complexity	Accuracy	Speed	Reagent Stability
Paper Chromatography	Low	Medium	Low	Low	Low
Thin Layer Chromatography	Low	Medium	Low	Medium	High
Enzymatic Testing	High	High	High	Medium	Low

Costs

The reagent and chromatography paper can be purchased for less than @ $A50, and allow analysis of up to 150 samples.

Up to 15 samples can be loaded onto a typical 20 x 20 cm piece of chromatography paper

Not including labour, cost of sample analysis could be as little as $A0.75!

Complexity

Some operator skill required in applying samples to the paper. To be effective, the method relies on the spots being as small as possible.

Some experience is required to confirm if the chromatogram has developed properly, or whether the results are meaningful.

Little sample pre-treatment is required, and no dilutions are necessary.

Accuracy

The method is qualitative only.

Absence of a malic acid spot does not necessarily confirm MLF is complete! A more accurate test will be required to confirm absence of malic acid.

Speed

Patience is required in applying sample to the paper. Allowing for two applications of the samples, and drying thoroughly in between and after, could take several minutes. If loading up to 18 samples, allow at least 15 minutes to complete the task.

TIP! Fill the capillary tube with only 1 mm of sample. This will prevent excessive spreading of the spot. The correct volume in the capillary is achieved by drawing up sample as normal, then holding a tissue to the tip to remove sample until only 1 mm remains.

Development of the chromatogram in the solvent bath can take up to 6 hours. Drying time can take several hours more.

Typically, the paper can be spotted early in the day, then left overnight to dry for interpretation the following morning.

Reagent Stability

The solvent mixture is very unstable, and will only keep for a few months if stored in the fridge.

TIP! Adding a few drops of formic acid, and drawing off any excess moisture can rejuvenate the mixture.

Overall Application and Suitability

This technique is suitable for small to medium wineries as a simple means of monitoring progress of MLF without the added expense of complex equipment.

Thin Layer Chromatography

Though similar to paper chromatography, this technique offers superior performance. Best results are obtained by using Schleischer and Schuell type TLC plates as described by Harris (1993). These plates are available from Vintessential Laboratories. The advantages of TLC over paper chromatography are,

Results are available within 15 minutes of spotting samples. Drying the plates is relatively fast.

Reagents are very stable. Provided the reagents are well cared for, at least 12 months shelf life can be expected.

Other attributes of this technique are similar to paper chromatography, namely,

Operating costs are similar. A 5cm x 7cm plate will allow up to 9 samples to be spotted. The technique is available in kit form for @ A$100, and @ 200 determinations can be performed.

Similar levels of expertise are required

The technique has the advantage over Enzymatic analysis in that the operator can see if MLF has been initiated by the appearance of a Lactic Acid spot.

Overall Application and Suitability

This technique is suitable for any winery but particularly for small to medium wineries as a simple means of monitoring progress of MLF without the added expense of complex equipment. Relatively quick results can be obtained, and the winemaker is able to determine the onset of MLF through to its near completion.

Enzymatic Analysis

This is a very accurate and reliable analysis that will allow quantification to 0.05 g/L.

Costs

The reagents are available in kit form and are relatively expensive. Typically, kits will allow 30 determinations, and cost under A$100. Along with the consumable costs, there are also other hidden costs prospective purchasers need to be aware of;

A standard recovery and blank measurement must always be performed with each set of samples. To ensure accuracy, duplicates should also be considered. Therefore, not all the reagents in a kit will be used exclusively for sample analysis.

A good quality Visible spectrophotometer is required for the analysis, and will cost several thousands of dollars. However, the instrument can then be used in conjunction with other kits for monitoring other parameters, such as sugars or lactic acid.

Good quality micro-pipettes are required for the analysis. At least two will be required, one for the 0 – 50 microliter range, and one for the 100 – 1000 microlitre range. Each will cost several hundred dollars.

The micro-pipettes will require periodical adjustment and re-calibration. A four-figure analytical balance is required for this or can be by an outside supplier.

The balance itself will require periodical (monthly) calibration checks, and certified weights are required for this. A set of 2 certified weights (eg a 1.000g low mass, and a 100.0000g high mass) are required. These weights themselves and the balance will also require servicing and re-certification (every 3 years for the balance, and every 5 years for the masses) from an accredited agency.

Complexity

Precise use of micro-pipettes and careful attention to method detail is required for the analysis to be successful.

A well-trained operator will be required to perform the testing, and also to maintain the micro-pipettes and the spectrophotometer in good working order.

Appropriate sample pre-treatment will be required. Turbid samples must be filtered, and dark coloured samples decolourised.

Accuracy

The technique has been around for several decades, and has proven to be very accurate and reliable.

The technique is very reliable for confirming completion of MLF. In our laboratory, we recommend a figure of <0.05 g/L malic acid as being a safe level.

Speed

The method is relatively fast, with a turn around time of @ 30 minutes (depending on the number of samples, and type of sample pre-treatment required).

Reagent Stability

Test kit reagents have a limited life, and must be stored cold. Typically, the NAD reagent will last for only 1 month.

Overall Application and Suitability

This technique is suitable for medium to large wineries. When performing a cost benefit analysis, wineries will need to consider not only the up front capital cost of the equipment, but also the extra costs for maintaining and calibrating the equipment. Reagent costs are also more expensive, and the expense of paying a suitably trained operator will also need to be considered.

Strategies to Maximise Value

In summary, the qualitative methods discussed are relatively inexpensive, provide useful information regarding progress of the MLF ferment, but cannot be used for confirming completion of MLF. Enzymatic analysis is expensive, but allows superior accuracy.

The use of an enzymatic analysis is probably beyond the budget of a small winery. TLC provides a very economical alternative, and can be utilised by the small winery to minimise costs significantly. Once malic acid can no longer be detected by TLC, then an accurate confirmation test can be done through an oenology laboratory. If the wine is in barrel, outside testing costs can be further minimised by preparing composites of, eg, 3 barrels. If malic acid is still detected in one of the composites, then the barrels can be tested individually to identify the laggard.

Similar protocols can be followed by medium to large sized wineries.