Describe the conditions necessary for the successful completion of a malo-lactic fermentation with a desirable flavour profile.
Malolactic fermentation (MLF) is a biological process that is initiated in the winemaking process to produce premium quality of wines. The conversion of L-Malic acid to L-Lactic acid by lactic acid bacteria (LAB) will have a direct effect on the wine produced in terms of quality. MLF increases microbial stabilization, sensory attributes of the wine by reducing the acidity level in the wine(Versari et al., 1999). Desirable attributes such as honey, vanilla and smoother taste in wine has been reported by several studies which is linked to the enzymatic activity of Lactic acid bacteria during Malolactic fermentation (Lopez et al., 2011).
During alcoholic fermentation, the wine becomes hostile for Lactic Acid Bacteria, due to the presence of high ethanol content, SO2 and other chemical residues released by yeasts. But at a controlled level, they could be made optimum for Malolactic fermentation. The species of Lactic acid bacteria assisting spontaneous fermentation belong to the Oenococcus, Leuconostoc, Lactobacillus genera. Although, Oenococcus oeni are the predominantly used LAB. They are considered to be ideal for Malolactic fermentation due to its specific oenological properties like reduced production of acetic acid, presence of enzymes that enhances the aroma and flavour characteristics, decreased risk of wine spoilage(CHRIS POWELL, 2005, Chalfan et al., 1977).
Wine fermentation is an important process carried out by microorganisms like yeasts and lactic acid bacteria. Malolactic Fermentation (MLF) is the enzymatic conversion of L-malic acid to L-lactic acid and Carbon dioxide by lactic acid bacteria. The importance of MLF in commercial winemaking process is due to the biological deacidification reaction by the wine lactic acid bacteria (LAB) (CHRIS POWELL, 2005). Malolactic Fermentation occurs after alcoholic transformation predominantly by Oenococcus oeni species. The bacterial activity during fermentation and its quality depends on the wine. Chemical and physical composition of the wine control the survival and growth lactic acid bacteria in wines(Toit, 2011).
Malolactic Fermentation has varied effects on wine flavour and aroma. MLF can either occur spontaneously in wine or by addition of starter cultures that are commercially available. With spontaneous MLF, there are different end results which is due to the native microorganisms that may occur immediately or few months after the completion of alcoholic fermentation. The indigenous microorganisms like lactic acid bacteria may start off from the vines and grape skin in the winery equipment. This leads to risks like increase in volatile acidity, delayed malolactic fermentation and development of unfavourable metabolites like biogenic amines. Biogenic amines affects the quality of wines and affect human health. These are primarily formed due to the decarboxylation of amino acids by Lactic acid bacteria(Solieri et al., 2010). This could be tackled by using a defined starter culture. In order to improve the quality of wine through MLF, wine makers prefer to inoculate grape musts or wine with lactic acid bacteria starter cultures. These are commercially available in freeze dried form or fresh liquid forms. Although few winemakers prefer to use their own starter cultures by growing strains of lactic acid bacteria in diluted grape juice or wine(Bisson, 2004).
Inoculated starter culture in Malolactic Fermentation provides better control on the timing of fermentation process and the organisms present. Bacterial starter culture is more difficult to maintain than the yeast starter culture, because the medium needs to be completely sterile and there is a high probability of undesirable lactic acid bacteria occurrence. Fermentation occurs faster under high inocula. The percent inoculum that is needed to be used should be 1-50% depending on the vigour of culture(Semon et al., 2001).
There are a number of risks associated with spontaneous malolactic fermentation. The timing of the process is sometimes uncontrolled and there is a higher threat of undesirable strains that could cause an off flavour in the wine. The timing of process is more certain for alcoholic fermentation than the MLF. This is due to the fastidious nature of bacteria occurring spontaneously in the fermentor(Solieri et al., 2010).
The use of starter culture instead of native culture has been a widespread winemaking practice. Inoculated Malolactic fermentation increases the quality and safety of wine produced. On the other hand Spontaneous Malolactic fermentation sometimes increases wine spoilage and produces toxic metabolites like biogenic amines(Solieri et al., 2010).
EFFECTS OF MALOLACTIC FERMENTATION:
The main effects of Malolactic fermentation are
The deacidification is due to the reduction of net concentration of carboxyl groups. The reduction in acidity is important for high acid wines and may not be desirable in wines already with lower acidity. There is a decrease in titratable acidity by 0.01 to 0.03 g/L because of hydrogen ion fixation(Kurane and Ghosh, 2012).
Various studies have reposted on the specific sensory changes that occurs in wine production due to the malolactic bacterial enzymatic activity. The sensory attributes are strain dependent. In addition to acidity reduction, flavour characteristics of wine after MLF can be buttery, nutty, earthy and fruity. The desired flavour profile in wine can occur in wine after MLF due to the removal of existing flavour compounds and production of new aromatic compounds with better sensory attribute. THE grape and yeast derived secondary metabolites get modified to end products with better desired flavour compounds from metabolism of sugar and amino acids(Bartowsky, 2005).
BASIC CONDITIONS NECESSARY FOR SUCCESSFUL MALOLACTIC FERMENTATION
pH: MLF strains grow at pH 2.9-3.0. If the wines have a very low pH after the primary fermentation, it is required to consider the reduction of acidity before MLF initiation. It is also important to consider the formation of biogenic amines. MLF cultures grow well even in pH higher than 3.5. Although the risk of off flavour and production of undesirable taste and aroma components increase as the pH increases. Therefore it is needed to adjust the acidity and pH before malolactic fermentation if the pH is more than 3.7. Malate is catabolised at pH 3.2 The enzymatic conversion of L-malic acid to L-lactic acid is faster at a pH 3.5 and the conversion rate is lower at lower pH values. The pH tolerance also relates to the strain variation and viability(Comfort, 2011).
Temperature: Temperature is an important factor for initiation or inhibition of Malolactic fermentation. The ambient temperature for the growth of malolactic bacteria is between 20 to 37°C. At a temperature below 15°C, the Malolactic bacteria are generally non-viable. Therefore warming down the wines to 18°C will allow the growth of lactic acid bacteria. Red wines at a higher temperature are suitable for MLF with general recommendations between 18 – 22°C. In traditional winemaking regions, MLF is initiated in spring where the wines are monitored for the formation of L-Lactic acid bacteria and thus warming down the fermentor to ensure timely completion of MLF (LOUBSER, 2005).
Free SO2 : It is important to maintain Sulfur dioxide levels in malolactic fermentation. Sulfur dioxide is added to prevent formation of undesirable lactic acid bacteria. Most species of Lactic acid bacteria are more sensitive to Sulfur dioxide than Saccharomyces species. The free form of SO2 is responsible for inhibited the undesirable lactic acid bacteria. The free SO2 is dependent on pH. The typical range of sulfite that is required needs to be added for white wines are 20-30 ppm and 30-50 ppm for red wines(LOUBSER, 2005).
Nutrient Composition: Lactic acid bacteria are more specific than yeasts and require additional micronutrients and growth factors. Availability of nutrients is essential for the malolactic conversion. Compared to yeast lactic acid bacteria also requires presence of additional amino acids. At the end of alcoholic fermentation, yeasts release amino acids and thus there is no limitation of amino acids during MLF. Autolysis of yeasts also increases micronutrient concentration. It has been reported that malolactic fermentation occurs better during pre-yeast fermentation or after the release of amino acids. However it limits the MLF when the lactic acid bacteria is added during the alcoholic fermentation. There are mixtures of lactic acid nutrients available commercially and it is required to time the addition of nutrients during the fermentation process(Torriani et al., 2011).
Oxygen: Oxygen stimulates MLF. It is one of the growth factor for lactic acid bacteria. However, the effect of oxygen varies with different species as few LAB get inhibited by oxygen. Oxygen level needs to be at a controlled level as higher oxygen level may sometimes lead to the production of acetic acid and undesirable end products. There have been occurrence of acetic acid accumulation in the wine when the Malolactic enzymatic activity occurs before the alcoholic fermentation. This is reported to occur alongside the aeration of juice or grape must(Comfort, 2011).
CO2 : Carbon dioxide stimulates MLF and helps with better vortex and mixing inside the fermentor. It also affects the buffering capacity of wine(Comfort, 2011).
ADDRESSING ISSUES OF HIGHER OR LOWER ACID CONTENT:
High level of acidity often occur in grape musts that are derived from grapes grown in cooler regions like France, Germany, cold regions of Australia. The wines made from these cold region vines, have improved flavour, microbial stability due to malolactic fermentation. Although sometimes the MLF may lead to excessive or lower acid content and sensory attributes(Massera et al., 2009). These issues can be addressed by two ways:
The initial Malic acid level in the must needs to be estimated which will help choose specific maloalcoholic strains. with higher malic acid levels, maloalcoholic yeast strains works better in controlling the acidity level(Massera et al., 2009).
There is another concern of development of excess diacetyl. The level of diacetyl increases by two metabolic pathways. One way they are formed is by metabolism of citric acid. Most wines have citric acid in them and some are produced even during primary fermentation. Hence by the end of Malolactic fermentation, the malic acid depletes and levels of diacetyl increase due to citric acid metabolism(Versari et al., 1999). During the citric acid metabolism, acetic acid is also produced that contributes to volatile acidity. One of the way to tackle excess diacetyl is by using malolactic cultures that have lower citrate metabolism like Lalvin MT01. Another way of which excess diacetyl gets produced is when the malolactic bacteria metabolizes malic acid. For example, in dry wines sugars like pentoses can be metabolized by lactic acid bacteria but cannot be yeast fermentable. Diacetyl is produced when these sugars are metabolized. This can again be prevented with regular monitoring of malic acid levels and stabilize the process when they are reduced(Massera et al., 2009).
During low acid conditions and high pH wines produced from warm climate vine, it is better to acidify the grape musts to favourable levels before primary fermentation. Malolactic fermentation of such low acid wines increases microbial stability and wine quality(LOUBSER, 2005).
Acidification of low acid musts is required for grapes and musts that are derived from vines of warm region. Adjusting pH and acidity before alcoholic fermentation is required. When Stronger acids like tartaric acid or DL- malic acid is used when the titratable acidity needs to be adjusted(Miller et al., 2011).
Deacidification of musts with higher acid content
Using engineered yeasts(Miller et al., 2011)
To obtain a desirable flavour profile through malolactic bacteria, various factors are needed to be considered and observed. Wine should be present at a temperature of more than 15°C and nutrients can be supplemented during the fermentation process. Because sometimes high solid content may halt the fermentation of wine. It is better to rely on a Malolactic inoculum rather than spontaneous initiation of Malolactic fermentation(Toit, 2011). The ethanol content should be regulated such that it does not inhibit the malolactic fermentation. It has been found that the lactic acid bacteria that grows in higher ethanol content will produce off flavours and unwanted end products in the wine. To summarise it is known that MLF can be stimulated by low or no use of free sulfur dioxide, warm temperatures, supplementing nutrients to the culture, lowering the ethanol content, adjusting the level of acid and pH range.
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