Questions 1. What can I do to enhance the occurrence of malolactic fermentation? 2. What are the applications of the new enzyme Lysozyme recently available in the European Union? 3. How do I know if my yeast is still fresh? 4. I hear lots of negative comments about mother tanks-are they true? 5. Can you ferment white wines (sauvignon blanc, chardonnay) at 10 ºC? 6. What is van der hofft's law? 7. What is the best way to add enzymes? 8. Does SO2 affect the activity of enzymes? 9. What is the affect of bentonite on enzymes? 10. What is the recommended dosage, temperature and contact time for enzymes? 11. What is the shelf life of enzymes? 12. Why are standard pectinases not recommended for red wines? 13. What is the best time to add enzymes for the maceration of red grapes? 14. What is lysozyme? 15. How do enzyme activities or units used by different suppliers compare? 16. What does GRAS stand for? 17. Why is direct inoculation with wine yeast better than inoculation after propagation? 18. How can one conduct partial MLF or stop spontaneous MLF? 19. What is the organoleptic impact of MLF on wine? 20. What are the ideal conditions for MLF in wine? 21. What are the nutritional needs of LAB? 22. What happens when MLF occurs in the presence of residual sugar? 23. When is the best time for MLF? Answers 1. What can I do to enhance the occurrence of malolactic fermentation? A number of factors contribute towards successful malolactic fermentations, and it should be remembered that these factors act synergistically. The contributing factors are pH, temperature, alcohol, SO2 and nutrient availability. The pH should be greater than 3,1. The temperature should ideally be between 20°C and 23°C and preferably no less than 18°C. The free SO2 should be no greater than 10 ppm, (recommended total SO2 for white wine 40 ppm and for red wine 70 ppm). Above 14%, alcohol becomes a significant inhibitor of malolactic fermentation. If using a starter culture, the best time of addition is at the end of alcoholic fermentation. Often there can be a nutrient deficiency, the most important of which is a nitrogen source. If it is to be added, the nutrient of choice is yeast extract, which supplies essential amino acids and short peptides, (the preferred nitrogen source), as well as vitamins and minerals. Many commercial companies recommend a starter culture procedure involving grape juice and nutrients before inoculating into the wine. << back to top 2. What are the applications of the new enzyme Lysozyme recently available in the European Union? Lysozyme is an enzyme (protein) derived from egg white and has been safely used in the food industry for a number of years. It acts to inhibit gram positive bacteria and has been demonstrated to inhibit lactic acid bacteria, including malolactic bacteria, in wine. It can be used to either delay or inhibit malolactic fermentation as may be required with some bottle fermented sparkling wines, or to inhibit bacterial spoilage in wine where reduced sulphur dioxide is used or wines with a high pH, under which conditions sulphur dioxide is less effective. << back to top 3. How do I know if my yeast is still fresh? The vacuum brick should still be hard (it will last for 3 months if it has just gone soft or after opening). Check the date stamp, it should be within the expiry date or within 2 years of the production date. Check also that the product has not been heated as this could have adverse effects on the quality. << back to top 4. I hear lots of negative comments about mother tanks-are they true? It is definitely better to use direct inoculation as this will help prevent sticking fermentations as well as ensuring the fermentation profile is better. Mother tanking reduces the sterol levels in the yeast cells, which can have very many negative effects on fermentation performance. << back to top 5. Can you ferment white wines (sauvignon blanc, chardonnay) at 10 ºC? Yes it is possible although very few wine yeasts can ferment at such a low temperature. In the Anchor range VIN 13 and N 96 will be able although we don't generally recommend it. The only advantage of fermenting very cold is increased aromatic ester production. Fermenting Chardonnay very cold will increase banana and pineapple aromas if a wine yeast capable of producing those esters are used. VIN 13 can produce such esters. In the case of Sauvignon Blanc we do not recommend such low temperatures. The typical varietal aroma of S. Blanc is expressed at slightly higher fermentation temperatures. We therefore recommend fermentation of S. Blanc with Anchor yeasts between 13 - 15°C. However if poor quality grapes are used and one has to rely on fermentation esters to produce a half decent wine then a colder temperature could possibly give a better result. In general the colder the fermentation the less mouth feel in a wine. This effect can be combated by leaving a little residual sugar - 4 g/L - in the wine. Sterile filtration is then recommended. << back to top 6. What is van der hofft's law? During fermentation between the temperatures of 10 - 40 ºC, a one-degree increase in temperature will result in a 10% rise in fermentation rate. << back to top 7. What is the best way to add enzymes? It is recommended that both liquid and granular enzymes must be diluted in cool water before addition. For a homogenous distribution the enzyme solution can be sprayed or dripped over the grapes during crushing. In the case of certain varieties settling enzymes can also be added directly into the settling tank if contact with grapes skins must be kept to a minimum. << back to top 8. Does SO2 affect the activity of enzymes? Yes it does but only in very high dosages. These dosages are usually way above the legal limit of SO2 concentrations permitted in wines and is therefore not a factor. However, care must be taken when making enzyme and SO2 additions at crushing. It is recommended that the SO2 addition is made before the enzyme addition to the grapes and that the SO2 is allowed to disperse, i.e. become diluted, before the addition of the enzyme. << back to top 9. What is the affect of bentonite on enzymes? Bentonite is negatively charged and absorbs positively charged molecules like proteins rather non-specifically. Enzymes are proteins and are absorbed by and therefore inactivated by bentonite. It is not recommended to settle with bentonite since it will remove the settling enzyme as well as valuable nitrogen components required by the yeast. However in the case of Botrytis infection settling with bentonite is inevitable since one must remove the laccase enzyme. If you need to add enzyme to a bentonite treated wine make sure the wine is completely racked off the bentonite lees before making any enzyme additions. << back to top 10. What is the recommended dosage, temperature and contact time for enzymes? It depends on the specific enzyme application, i.e. settling, white skin contact, red skin contact etc. The three parameters influence each other. For instance in the case of white skin contact, the higher the temperature – the shorter the contact time and the lower the dosage. Different enzymes have different recommended dosages and instructions must be followed very carefully for optimum results. More is not necessarily better and one can over macerate. In the case of white musts over maceration is characterised by very clear juice at the top of the tank, very compact lees at the bottom and a meter or two (depending on the tank size) of “fluff” that is impossible to settle. This fluff is little pieces of grape skin that is broken into small pieces. It is only necessary to break skin cells open to obtain more juice and flavours, not to break it into small little pieces. << back to top 11. What is the shelf life of enzymes? Granulated enzymes are very stable if stored under the recommended conditions. Their shelf life is usually indicated as 3 years but it is actually longer if they remain un-openend. Liquid enzymes are less stable than granulated enzymes but if stored under the correct conditions the activity loss is minor and can usually be corrected with an increase in dosage. << back to top 12. Why are standard pectinases not recommended for red wines? Standard pectinases not specific for red skin contact, contain an activity known as anthocyanase. This is the generic name and the actual enzymes are different types of glycosidases. Glycosidases remove sugar molecules from more complex molecules from which anthocyanin is one. Anthocyanins are stabilised by the sugar components bound to them and when removed the anthocyanin becomes unstable and colourless. Standard pectinase preparations contain these activities because A. niger species usually produces a “cocktail” of enzymes. In the case of red skin contact enzymes a specific fungus producing very low levels of this activity must be used. << back to top 13. What is the best time to add enzymes for the maceration of red grapes? Enzymes should be added as early as possible, preferably at the time of crushing since this is the beginning of the colour extraction phase of red wine maceration. Most colour extraction takes place at the beginning of fermentation whereas tannin extraction takes place towards the end of fermentation and during post fermentation maceration. << back to top 14. What is lysozyme? Lysozyme is an enzyme extracted from egg whites. It kills gram-positive bacteria that include lactic acid bacteria (LAB). It is not pH dependent like SO2 and SO2 also only inhibits LAB without killing them. Lysozyme can be used to control the onset of malo-lactic fermentation (MLF) as well as to control wine spoilage. It will therefore increase microbial stability of wines and allow a reduction of SO2 usage. << back to top 15. How do enzyme activities or units used by different suppliers compare? They don’t compare at all. Companies use different substrates to measure activities as well as express the activities in different units. The only way to compare products is to do trials. << back to top 16. What does GRAS stand for? GRAS stands for Generally Regarded As Safe. This statement applies to microorganisms used in the food industry. Wine enzymes may only be produced from GRAS organisms. << back to top 17. Why is direct inoculation with wine yeast better than inoculation after propagation? When yeast is grown in the presence of alcohol and in the absence of oxygen, the sterol content in the cells decrease considerably making the yeast more vulnerable to alcohol toxicity. In a yeast plant the yeasts are grown in the absence of alcohol and in the presence of a massive amount of oxygen thereby generating lots of sterols and long chained fatty acids necessary for alcohol tolerance. Direct inoculation therefore reduces the risk of lagging and stuck fermentations. << back to top 18. How can one conduct partial MLF or stop spontaneous MLF? It is quite difficult to stop spontaneous MLF once it has started. If partial MLF is desired on a wine it is best to blend wine that has completed MLF with a wine that has not undergone MLF. In order to prevent MLF completely one can use several methods either separately or in combination. These factors are low temperatures (below 18°C), sulphur dioxide (inhibitory to LAB) and lysozyme (kills LAB). << back to top 19. What is the organoleptic impact of MLF on wine? Malic acid is converted to lactic acid that is less acidic than the former. This loss in acidity and accompanied increased pH has a profound effect on taste. During MLF new aromatic compounds are formed that can change the aroma profile of a wine and increase mouth-feel. MLF is also characterised by the slight decrease in upfront fruit intensity and freshness of a wine. Generally a wine that has been through MLF can be well balanced with a complex aromatic profile. MLF is only suited to certain white grape varieties. << back to top 20. What are the ideal conditions for MLF in wine? -Alcohol below 14% -Temperature above 15°C -pH above 3.0 -Total SO2 below 60 ppm -Free SO2 below 10 ppm -Minimum number of cells: 106 CFU / ml << back to top 21. What are the nutritional needs of LAB? Lactic acid bacteria like other microorganisms need a carbon source and a nitrogen source. They can utilise various carbohydrates and only organic nitrogen like amino acids. It is impossible for MLF to utilise inorganic nitrogen like di-ammonium phosphate. Several growth factors and vitamins like thiamine and biotin are also required. The use of an adapted MLF nutrient – based on inactive yeast – will supply these substances in sufficient quantities to conduct a healthy MLF. << back to top 22. What happens when MLF occurs in the presence of residual sugar? If LAB starts to grow before the end of alcoholic fermentation they utilise glucose and fructose instead of malic acid. The result is different end products leading to an increase in “buttery flavour” and volatile acidity. << back to top 23. When is the best time for MLF? The best time for MLF is right after alcoholic fermentation. Wine temperature will still be elevated - and therefore very suitable for MLF - due to heat generated by the yeast during fermentation. After completion of fermentation various cellular components “leak” into the wine through the weak yeast cell membrane and cell wall. These components include amino acids and nucleotides that can serve as organic nitrogen for LAB. It has also been proved that small quantities of CO2 help LAB develop. << back to top