Co-inoculation | Vintessential Wine Laboratories

Introduction

Commercial strains of malolactic bacterial cultures have been available for many years. Until recently, these cultures were added after the completion of the alcoholic (yeast) fermentation. New research has enabled the development of strains of bacteria that can be added at the same time that yeast is added (co-inoculation). The advantages of co-inoculation are many, and these will be considered in comparison to no inoculation and inoculation after alcoholic fermentation, in the following article.

Malolactic fermentation
Malolactic fermentation (MLF) is widely employed because it metabolises the microbially-unstable malic acid found in many wines, particularly red wines. Almost all finished wine (apart from a small amount of preservative-free wine on the market) is protected from microbial growth by the addition of sulfur dioxide. This preservative inhibits the lactic acid bacteria (LAB) in wine that can ferment malic acid. However in red wines there are generally lower levels of sulfur dioxide, and these levels decrease with time in the bottle. Also filtration of red wines does not always remove all bacterial cells from the wine. These are some of the main reasons why most, if not all winemakers want their red wines to undergo MLF.

MLF is not always an easy fermentation, especially when compared to yeast fermentation. Much work has been done over recent decades to produce commercial bacterial products that not only make MLF simpler for the winemaker, but also make the fermentation go through to completion.

No inoculation for MLF
Alcoholic fermentation (AF) by yeast has been widely studied and is well understood, compared to malolactic fermentation (MLF) by bacteria. Non-inoculated MLF has, of course, been occurring since wine has been made. The bacterial species occurring in MLF include Oenococcus, Lactobacillus and Pediococcus, however, results are not guaranteed by using non-inoculation. The resulting wine can also be spoiled by other organisms that may grow if the non-inoculated MLF is starting very slowly and proceeding very slowly, whilst being offered little protection from the presence of sulfur dioxide.

Issues that can occur in non-inoculated MLFs include:
-delayed onset of MLF
-lengthy MLF, going on for many months
-production of ‘off’ flavours
-production of biogenic amines such as histamine
-increased volatile acidity
-co-growth of spoilage organisms such as Brettanomyces

The main parameters in wine that affect the performance of the different bacterial species include pH, alcohol, temperature and the presence of nutrients. Different species of MLF bacteria differ in their tolerance to these conditions, and if the conditions in a wine have two or more of these parameters at unfavourable levels, MLF can be exceedingly difficult to complete, as many winemakers would be aware.

As a consulting laboratory, we work with many hundreds of wineries and although we receive a number of enquiries about yeast fermentation each year, the problems with MLF are far more frequent and usually more difficult to assist with.

Inoculation after alcoholic fermentation
The use of commercial bacterial cultures carefully selected for MLF and added to the wine after AF is now very common. This innovation has helped overcome many of the problems that occurred with non-inoculated MLF. The main bacteria that has been used in these commercial products is Oenococcus oeni (formerly known as Leuconostoc oenos). The development of freeze dried bacterial cultures and simple ways of adding these cultures to the wine have also enabled a quite fuss free way to get MLF done.

Some of the improvements offered by commercial MLF cultures include:
-better control of start time and duration of MLF
-lessening of biogenic amine production
-reduced time that wine is unprotected by sulfite and so less potential for spoilage by other organisms
-reduced incidence of excessive volatile acidity
-enhanced flavour profile

However there are still issues with using commercial MLF bacteria. This is mostly due to the fact that one of the main inhibitors of bacterial activity, and therefor MLF, is present in large quantities, just when it is not desired – alcohol.

Some of the issues that can occur with inoculation post-AF include:
-slow MLF due to high ethanol contents
-the need to warm the wine to ensure MLF proceeds well, with concomitant cost

Co-inoculation with alcoholic fermentation
Much research has been done recently on adding MLF bacteria at the start of AF. It is now to the stage where several commercial products are well established and much experience in the winery has been gained using this process. By adding these bacterial products at the start of AF the inclusion of species other than Oenococcus has also been enabled, thus giving winemakers the opportunity to focus on other aspects of MLF, such as improving flavour development during the fermentation.

One species that has been found to be particularly exciting is Lactobacillus plantarum. Research done with these bacteria has found that key aroma compounds such as esters and terpenes have increased concentrations and provide an extra flavour profile to the resulting wine. As mentioned, this is now accepted practice and many winemakers are using these products to not only complete MLF more easily, but also enhance the flavour profile of their wine.

Some key advantages of using co-inoculation are:

-lower levels of the inhibitory alcohol are present at the start of MLF
-no need to apply external heating to the ferment due to the heat generated by the yeast fermentation
-faster completion of MLF, and thus the wine can have sulfite added earlier and so reduce the potential for the growth of spoilage organisms
-by adding the bacteria at the start of the yeast fermentation a nutrient rich environment is encountered
-potential to use non Oenococcus strains such as Lactobacillus plantarum to provide enhanced flavour profiles

Conclusion
The commercial use of bacterial strains for malolactic fermentation (MLF) has increased markedly in the last couple of decades. The advent of these commercial products has greatly increased the reliability of MLF. Further developments have recently taken place in the use of MLF bacteria applied at the start of alcoholic fermentation. These improvements, along with the use of innovative species such as Lactobacillus plantarum, have further enhanced the experience of winemakers. This important, but historically difficult fermentation now has another enhanced technique that winemakers can employ in their quest for making better wines.

Greg Howell is the founder and Managing Director of Vintessential Laboratories. He can be contacted by email on [email protected]/. More articles on related topics are available on the Vintessential website: www.vintessential.com.au/resources/articles/