The History and Science of Malolactic Fermentation

The History

Brad Webb with Ambassador Zellerbach (Photo courtesy of Hanzell Vineyards)

Brad Webb had landed the dream job. It was 1956, and he’d just been appointed winemaker at a new winery, Hanzell, founded by James D. Zellerbach, the wealthy US ambassador to Italy. Zellerbach had spared no expense in pursuing his dream of making classically styled Chardonnay and Pinot Noir to rival the wines of Burgundy. Webb had at his disposal an array of shiny new tanks, casks, and barrels. But there was a problem. The early Pinot Noir that he made in the new facility looked promising, but it wouldn’t do malolactic fermentation (MLF), the conversion of malic acid to the softer lactic acid that usually takes place after alcoholic fermentation.

At the time, a growing awareness of the importance of MLF for high-quality red wines was developing, although a lot of mystery still surrounded this second fermentation. Sometimes it started of its own accord; sometimes it didn’t. Sometimes it finished quickly and easily; other times it dragged on, only to start again at a later stage. One of the inside jokes among winemakers goes something like this: How do you start MLF? Bottle the wine.

The first mention of MLF is thought to be in a 1837 book by Freiherr von Babo. He described a second fermentation occurring in some wines during springtime, when temperatures began to rise, resulting in the release of CO2 and renewed turbidity in the wine. Then, in 1866, the celebrated scientist Louis Pasteur isolated bacteria from wine for the first time. However, he considered all bacteria in wine to be spoilage organisms. A breakthrough came in 1891 when Hermann Müller-Thurgau (yes, that Müller-Thurgau—the grape is named after him) postulated that acid reduction could be because of bacterial activity. This was a bold suggestion; at the time, this change in acid level was generally attributed to tartaric acid precipitation. Müller-Thurgau went on to do valuable work over the next couple of decades in collaboration with a fellow Swiss researcher called Osterwalder.

In 1939, the famous French wine scientist Émile Peynaud wrote an important paper on the role of malic acid in the musts and wines of Bordeaux, pointing out that the absence of MLF was a quality-limiting factor in these wines. “Not only is the acid makeup of the wine completely changed,” he stated, “but [MLF] has an impact on the perfume of these wines and even diminishes the intensity of the color and changes its shade. It is not exaggerating to say that without malolactic fermentation, there would hardly be any great reds of Bordeaux.”

While yeast cultures were starting to become available, no one had successfully cultured the lactic acid bacteria (LAB) responsible for the conversion of malic to lactic acid, with its corresponding softening of acidity. Webb had to resort to experimentation to try to get this fermentation to start. Could the reluctance of his ferments to do MLF be because of the lack of suitable bacterial inoculum in the winery? He tried introducing wine that was undergoing MLF into his tanks, but it didn’t work. Baffled, he turned to John Ingraham, a scientist at UC Davis who had an interest in bacteria. Webb offered his winery as an experimental setting where Ingraham could do trials—after all, in almost all other wineries, MLF took place. So it should be of interest to study a winery where such a fermentation had never occurred. Ingraham was intrigued and willing to use Hanzell as a negative control.

Ingraham had been busy at work, and the timing was good. Research on LAB, which are also important in the production of other foodstuffs, had been flourishing ever since the serendipitous discovery by a dental researcher in Chicago that adding tomato juice to MRS agar, the bacterial growth medium commonly used for making microbial cultures, made these previously tricky bacteria easier to grow in the laboratory. It turns out that tomato juice contains pantothenic acid, a key growth factor for these bugs. Ingraham had isolated 50 strains of LAB from samples of dry wines and lees from California wineries. Of these, with the help of Webb, he selected one that showed the most promising characteristics. They named it ML34. Although this was kept secret at the time, it came from a large redwood tank of Barbera in the Louis Martini winery in Napa.

In 1959, the two took ML34 to Hanzell to study it in winery conditions. After some trials on a small scale, to their great excitement, they got ML34 to carry out a malolactic fermentation in the winery. To their knowledge, they were the first to do this. But, as so often happens in science, other groups had been working on the same problem elsewhere. In France, Peynaud had achieved this with his colleague Domercq, and a short time before, a Portuguese group had also been successful. But this doesn’t take anything away from the achievements of Webb and Ingraham, who published the results of their work in 1960.

Original ML34 Test Tube at Hanzell (Photo courtesy of Hanzell Vineyards)

How Malolactic Fermentation Works

Malic acid is an organic compound with the formula C₄H₆O_5. It is found in all fruit but is most associated with green apples.

During MLF, the LAB convert malic acid to lactic acid, which is softer tasting and less powerful. There is a pH shift and a loss of acidity, the degree of which will depend on how much malic acid was present in the grapes in the first place. MLF happens in almost all red wines, and the choice of whether or not to let it occur in whites is usually a style decision on the part of the winemaker.

But there is a lot more to MLF than this acidic conversion. Just as yeasts have a significant sensory impact above and beyond simply converting sugar to alcohol, LAB change the flavor of wine in ways that are only now becoming appreciated as scientists take a closer look at the activity of these microbes. The sensory impact of LAB can be both positive and negative, and it depends largely on the strain of bacteria that is doing the fermentation, the presence of certain substrates in the wine, and the conditions under which the fermentation takes place.

Grape juice and wine are hostile environments for microbes. On the one hand, grape juice has what microbes really want: sugar. It’s just that it has rather too much of it, around 200 grams per liter. This creates a strong osmotic pressure that threatens to suck all of the water out of the bugs. Grape juice also has too much acidity. Bacteria like to grow in a higher pH medium, with an optimum level around 6 or 7. Grape juice and wine have a pH of about 3 to 3.8, which is quite acidic. Initially, there will be numerous species of yeasts present in grape juice, as well as many strains of bacteria. As fermentation kicks in, however, the alcohol level begins to rise, and the diversity of microbes is much reduced. After four days or so, only one species remains in any significant numbers: Saccharomyces cerevisiae. This comes in many strains, and even wild or native ferments will be carried out largely by this species. There are just a few species of LAB that can cope with the unfavorable environment, and by the time they get to carry out their second fermentation, which normally begins as alcoholic fermentation is finishing, they must contend with extra problems. These include high alcohol, possibly the presence of some sulfur dioxide, and a medium that has been stripped of many vital nutrients by the yeasts.

Bacteria are so tiny that they can be pretty hard to classify, and it’s only with the advent of modern DNA techniques that things have become clearer. The first distinction is between the shape of the bacteria: those that look like rods are called rods, and those that look like little spheres are called cocci. These are further classified depending on the way they carry out metabolism. In this way, LAB can be classified as heterofermentative or homofermentative, depending on how they ferment sugars. The distinction here is that the former ferment sugar to produce lactic acid, acetic acid, and ethanol, while the latter ferment sugar to produce only lactic acid.

Getting Technical

The four genera (a genus is the level of classification just above species) of LAB found in wine are Oenococcus, Leuconostoc, Lactobacillus, and Pediococcus. Of these, there’s one species, Oenococcus oeni, that is of particular interest. Winemakers want this species to carry out the second fermentation, as it produces the best results, and it usually does. Oenococcus oeni is pretty resistant to the hostile conditions found in wines just after alcoholic fermentation has finished—much more so than the other three genera, which tend to grow only when the pH is a little higher.

The LAB feed off any sugars that remain in the wine after the yeasts have finished (yeasts leave a bit of hexose and pentose sugars) and grow in number. The bacteria need less than 1 gram per liter of sugars to create a biomass sufficient enough to carry out MLF. Alongside this activity, they are also able to convert malic acid to lactic, but it’s worth emphasising that this is just one of their metabolic activities.

During the course of their growth, LAB are able to secrete a range of flavor compounds into the wine. This is where bacteria have the potential for enhancing quality or impacting it negatively. Indeed, it is really instructive to taste experimental wines inoculated with different strains of cultured malolactic bacteria. This sort of comparison shows the degree to which MLF can change the taste of wine in addition to modifying its acidity. But there’s very little written on the subject, and most winemakers allow MLF to happen spontaneously, trusting that they are going to get a decent strain of malolactic bacteria.

LAB produce acetic acid from metabolizing sugars, increasing volatile acidity. The amount that volatile acidity increases depends on how much sugar they metabolize, so this is a potential concern when MLF starts before the yeasts have finished their job. Again, this VA increase is quite strain dependent. As well as degrading malic acid, certain strains of LAB can degrade tartaric acid, too. Pasteur called this the tourne disease, and it’s a serious issue. Fortunately, very few strains can do this.

One of the most well-known sensory impacts of LAB is the production of diacetyl (2,3-butanedione). It’s formed by LAB from citric acid and has an odor detection threshold of 0.2 milligram per liter in white wines and 2.8 milligram per liter in reds (this higher threshold is because there are so many other strong flavors in red wine that it’s harder to pick up the dactyl). Diacetyl has a distinctive buttery, creamy character that, at low levels, can be attractive. But higher levels of diacetyl aren’t pleasant and can be considered a fault, and in some circumstances, any detectable diacetyl is undesirable. The factors favoring diacetyl production are the presence of oxygen, high concentrations of citric acid and sugar, temperatures below 18 °C, and the removal of yeast cells before MLF. The levels can be reduced by the presence of viable yeast cells and the addition of sulfur dioxide. Diacetyl can react with cysteine, an amino acid-containing sulfur, to produce thiazole, which smells of toast, popcorn, and hazelnut. The actual level of diacetyl isn’t closely correlated with the extent of MLF, however. Its production is strain dependent, and dependent on the precursors present as well. It’s entirely possible that a part-MLF Chardonnay could show more buttery flavors than one with full MLF. 

The production of volatile sulfur compounds (VSCs) is another way LAB impacts wine flavor. These are produced by the metabolism of the sulfur-containing amino acids cysteine and methionine, and the VSCs that result can be good or bad, depending on the context. These are the compounds implicated in reduction problems in wines: sulfites, disulfides, thioesters, and mercaptans (thiols).

The bitter-tasting compound Acrolein is produced by some LAB strains by the degradation of glycerol. It’s undesirable at any level, but thankfully, only a few strains produce it.

An often-discussed byproduct of MLF is the formation of biogenic amines. All fermented products contain them, but malolactic bacteria are capable of producing reasonably high levels. They are formed by the decarboxylation of amino acids, and the major ones found in wine are histamine, tyramine, putrescine, and phenylethylamine. They can have a range of effects on people sensitive to them, including headaches, breathing difficulties, hypertension or hypotension, allergic reactions, and palpitations. People differ in their sensitivity to them, but their presence in wine is undesirable. Not all strains of LAB are able to decarboxylate amino acids. The higher the wine’s pH, the more complex the range of bacterial species that will grow in it. As a result, there will usually be higher levels of biogenic amines. White wines, then, which usually have a lower pH, tend to have lower levels of biogenic amines. Although sulfites are often blamed for allergic reactions to wine, it’s much more likely to be the biogenic amines that are responsible, although this hasn’t been proven conclusively. Using selected strains of LAB to inoculate for MLF is one way to reduce the risk of biogenic amines in wines. Currently, there are no regulations for biogenic amine levels in wine, but this could change.

But biogenic amines aren’t the worst thing produced by certain strains of bacteria. Ethyl carbamate is a carcinogen found in many foods and drinks, and it’s formed through reactions between alcohol and a precursor such as citrulline, urea, or carbamoyl phosphate. The main contributor to ethyl carbamate levels in wine is urea formed by yeasts from the degradation of arginine, but even after alcoholic fermentation, some arginine (0.1 to 2.3 grams per liter) remains in the wine, and LAB can produce citrulline as an intermediate in the degradation process of arginine. The USA has regulations for maximum levels of ethyl carbamate in wine, set at 15 micrograms per liter. Canada allows up to 30 micrograms per liter, and the EU has no uniform maximum level. Typically, wine contains around 10 micrograms per liter, while fortified wines contain around 60, but these levels can vary.

Some strains of LAB are thought to have glycosidase activity, which has a positive effect. Many of the flavor molecules in grape juice are in a chemical state where they need to be converted during fermentation to be active. A glycosidase is an enzyme that removes sugar groups, and in this case it can hydrolyse sugar-bound monoterpenes to release them as volatile aromatic monoterpenes. There is also evidence that LAB are able to synthesize esters, which are fruity-smelling compounds, but this needs to be further verified.

LAB are able to remove green flavors from wine. The reduction of vegetative or grassy aromas that can occur during MLF is thought to happen through the metabolism of aldehydes such as hexenal, which contribute to these green flavors (along with methoxypyrazines). LAB are also thought to be able to improve the body of a wine, through, for example, the production of polyols and polysaccharides.

Though MLF affects flavor in a variety of ways, the most significant occurs through its impact on acidity. It usually increases pH (makes the wine less acidic) by 0.1 to 0.3 units and reduces TA by 1 to 3 grams per liter.

Conclusion

Most MLFs are spontaneous, but it is becoming increasingly common to inoculate with bacterial cultures. These come in different forms. There are freeze-dried cultures that need to be reawakened, making a starter culture with which to inoculate the wine, and there are now active dried cultures as well. Cultured bacteria are a bit fussier than cultured yeasts and need to be handled carefully. Previously, it was thought that the only safe time to inoculate was after alcoholic fermentation had completed. This was because of the risk of volatile acidity increasing (if the bacteria are eating lots of sugar), and the risk of an incompatibility between the bacteria and yeasts, causing fermentation to stick. Now, however, co-inoculation with compatible strains of bacteria and yeasts is commonplace. The advantage is that as long as the two microbe strains work well together, the result is fruitier wines (especially reds), in part because any diacetyl produced by the bacteria is used by the yeasts. As such, co-inoculation is particularly advised for less expensive, fruit-forward red wines, with the added bonus of no risk period between the end of alcoholic fermentation and the beginning of MLF.

Clearly, there’s a lot more to lactic acid bacteria than simply the conversion of malic to lactic acids. These microbes have an important role to play in winemaking, one that we are only just beginning to understand.