The term fermentation is nowadays understood to describe almost any process which enlists the aid of micro-organisms as a means of preparing useful products, very often difficult to obtain by any other method, from more readily available materials. It therefore embraces the production of such diverse and commercially important substances as alcohol, acetone, acetic acid (vinegar), citric acid, n-butanol, numerous antibiotics (e.g. penicillin and aureomycin) and so on. In winemaking, however, fermentation is generally assumed to refer only to alcoholic fermentation, i.e. the process by which yeast converts the sugar in a must into alcohol and carbon dioxide together with minor amounts of various byproducts, and thereby creates wine. It follows from this definition that successful winemaking is fundamentally dependent upon conducting a sound fermentation and that a clear understanding of at least the elementary principles of fermentation is obviously a great aid towards improving wine quality.
In the past, a great aura of mystery surrounded the process of fermentation. As long ago as 1810, the French chemist Gay-Lussac correctly advanced the following equation to account for the formation of alcohol and carbon dioxide from sugar during fermentation, but at that time no satisfactory explanation of reason for this occurrence had been suggested. Various improbable theories had been proposed to explain this phenomenon, but because yeasts were then not considered to have any significance in this respect no further progress was made for many years.
C6H1206 - 2CH3CH2OH + 2C02
Glucose
or Fructose Ethyl Alcohol Carbon Dioxide
Following the pioneering work of Pasteur in the latter part of the nineteenth century, the fact that yeasts were responsible for causing mentation gradually won general acceptance. It then became [ear that the Gay-Lussac equation, as it is called, merely repressed an overall picture of alcoholic fermentation. Thus, it cannot account for the formation of small amounts of certain by-products which are invariably produced during a normal fermentation in addition to alcohol and carbon dioxide. This discovery naturally stimulated a great deal of research work in this field designed to elucidate the mechanism by which fermentation proceeds.
Eventually, as a result of much sustained scientific investigation, was shown that fermentation, far from being a simple process, proceeds by a complex series of step-wise inter-related chemical reactions initiated and controlled by substances called enzymes which are secreted by the individual yeast cells. Enzymes are dual special proteins which can promote or catalyze specific chemical reactions in living cells without undergoing any permanent change themselves. In the case of yeast, a group of enzymes collectively known as the zymase complex is responsible for controlling fermentation.
The fact that enzymes secreted by the yeast cells rather than the actual living cells themselves are the active agents in fermentation as proved shortly before 1900 when Buchner showed that an extract prepared from yeast containing neither living nor dead cells but only the enzymes of zimase complex (and other water-soluble matter) could induce fermentation on its own. Each individual enzyme in this enzyme complex was also found to govern one and only one of the intermediate stages of fermentation. Once its task is been completed, another enzyme takes over and so on, the process continuing in such a way that each sugar molecule under- goes a progressive series of enzymic reactions which finally result in its degradation to alcohol and carbon dioxide with the release of a rail amount of metabolic energy.
The introduction of modern techniques such as radio-active icier studies provided further valuable information regarding the mechanism of fermentation. Consequently, the chemistry of this process is now know in considerable detail. Although few winemakers are likely to want a more detailed exposition of this particular subject, this chart showing the general scheme of the principal chemical reactions occurring during fermentation and accounting for the formation of the important by-product glycerol (glycerine) has been included for the sake of completeness.
One interesting feature of fermentation worth noting from this tart is that the majority of the intermediate reactions involve the inter-conversion of complex organic phosphates. It is for this reason at phosphates are such important yeast nutrients.
From these remarks, it may be concluded by way of a summary at alcoholic fermentation takes place through a long complex series of chemical reactions which result in the eventual degradation the sugar in the must into alcohol, carbon dioxide and a number by-products, but that in essence the overall reaction can be pressed in simpler terms by means of the Gay-Lussac equation, n this latter basis, it can be calculated that theoretically 100 gms. sugar should yield 51.1 gms. alcohol and 48.9 gms. carbon dioxide, practice, however, only about 48 gms. alcohol and 47.5 gms. carbon dioxide are actually obtained from this weight of sugar. A number of factors are responsible for this difference, but usually the losses of alcohol sustained during fermentation can be attributed to the fact that a small amount of sugar is used up in the formation of by-products and a little more sugar is metabolised completely to carbon dioxide and water and/or converted into storage carbohydrate glycogen by the yeast. Further small losses of alcohol also occur by its evaporation or entrainment in the gas evolved during fermentation. It therefore follows that secondary factors such as temperature, rate of fermentation, availability of oxygen and so on which influence these primary factors must also have some bearing on the final yield of alcohol. On the average, however, the winemaker may reasonably expect to obtain 90%-95% of the theoretical yield of alcohol from a given weight of sugar. The potential alcohol tables in the chapter on the hydrometer have accordingly been calculated on this assumption.
In addition to the principal products, alcohol and carbon dioxide, a number of by-products are also always formed during a normal alcoholic fermentation, notably the trihydric alcohol glycerol (glycerine) which is usually present in wines to the extent of 1% - 2%. Most of this glycerol is produced near the beginning of the fermentation. The presence of a small amount of acetaldehyde, a substance formed by one of the last enzymatic reactions in the complete fermentation series, is essential if the overall reaction is to yield alcohol. Acetaldehyde is important in this respect because it acts as a hydrogen acceptor for an oxidation-reduction enzyme system. The reduced enzyme is converted back into its oxidised form by the acetaldehyde which is consequently itself reduced to alcohol. During the early stages of fermentation, no acetaldehyde is present so that the whole series of enzymatic reactions leading finally to the formation of alcohol is delayed until sufficient acetaldehyde is produced to allow these reactions to proceed. In the meantime, another substance called dihydroxyacetone acts as an alternative hydrogen acceptor with the result that it becomes reduced to glycerol. Once sufficient acetaldehyde has accumulated, the latter reaction is discontinued and fermentation proceeds normally to yield alcohol. Subsequently, little more glycerol is formed.
It follows from this discussion that any substance which reacts with acetaldehyde to form an inactive complex would promote the production of glycerol by extending the duration of the dihydroxyacetone reduction reaction. Indeed, in extreme cases, the fermentation could even be adjusted to yield glycerol rather than alcohol as the major product, and this was in fact practised during the last war to augment glycerol supplies from other sources. One substance which complexes acetaldehyde in this way is sulphite. The practice of sulphiting the must prior to fermentation thus almost invariably leads to wines with a fairly high glycerol content. Sulphiting after racking will also increase the glycerol content of a wine if fermentation is subsequently renewed. Any acetaldehyde already in the wine at the time of racking will be bound by the sulphite and further glycerol will be produced until the deficit of acetaldehyde is made up and fermentation again gets under way. Other factors which favour glycerol formation include lower fermentation temperatures, and a higher proportion of tartaric acid in the must, but the strain of yeast used to conduct the fermentation also seems to have an important influence in this respect.
Pure glycerol is a dense, viscous, rather oily liquid (specific gravity 1.260) with a distinctly sweet taste. In view of the fact that wine usually contains some l%-2% of this substance, its effects on the flavour and general quality of the wine obviously cannot be ignored. Apart from adding slightly to the sweetness of a wine, glycerol also adds body and helps to create a smoother flavour by virtue of its oleaginous properties. Indeed, the addition of glycerol to wines containing too much tannin or too much acid is a recognised means of reducing their harshness or tartness to a more palatable level. Wines which have been produced from grapes infected by the mould Botrytis Cinerea usually have an exceptionally high glycerol content. In this rather special case, some of the glycerol content. In this rather special case, some of the glycerol is formed in the grapes as a result of the fungal attack and the remainder is subsequently produced as a fermentation by-product in the normal way.
A second important by-product of fermentation is fusel oil. This material is not a single substance but a mixture of higher alcohols, mainly isomeric amyl alcohols. Unfortified wines normally contain less than 0.5% of these constituents. Most of this fusel oil is formed by the de-amination of amino-acids, especially leucine and isoleucine, during the nitrogenous metabolism of the yeast. It has now been established that by no means all the fusel oil found in wines except in very low concentrations, otherwise its powerful aroma and rather harsh flavour, not to mention its poisonous properties, may prove objectionable. The addition of readily available sources of nitrogen, e.g. ammonium salts, to the must prior to fermentation helps to decrease the amount of fusel oil formed. The use of yeast nutrients containing ammonium phosphate thus will automatically aid in keeping fusel oil production at a low level.
The only other notable by-product of fermentation is succinic acid which is formed in quantities amounting to about 1% of the alcohol, e.g. a wine containing 15% alcohol would also contain about 0.15% succinic acid. This substance is important in wines because it helps to promote vinous character. It is derived at least in part by de-amination of the amino-acid glutamic acid.
Mention may also be made of the fact that very small amounts of acetaldehyde, 2 : 3-butylene glycol, acetyll methylcarbinol, diacetyl, acetic acid, lactic acid, methyl alcohol (from pectin degradation), hydroxymethylfurfural (in heated wines) and so on are all recognised by-products of fermentation. With the exception of acetaldehyde which has a strong aroma and may therefore add to the bouquet of a wine, these substances make little difference to the quality of a wine, mainly because they are present in such small quantities. Admittedly, lactic acid can be the principal acid present in some red wines, but in such cases it has been produced by a malolactic fermentation and cannot therefore be classified as a true yeast fermentation by-product. Similar remarks apply to acetic acid formed by bacterial infection.
Studies of fermentation other than from the chemical standpoint have shown that it can be divided into three principal phases of activity called the lag phase, the primary fermentation and the secondary fermentation respectively. The lag phase may be defined as the induction period or time lapse which is observed between the inoculation of the must with the yeast and the first visible signs of fermentation. The duration of this lag phase can vary from a few hours to several days, for it is dependent upon a number of factors, one of the more important of which is the initial concentration of yeast in the must. Fermentation normally cannot commence until the must contains about 30 million cells per fluid ounce. Because it is rare to find such a high initial concentration of yeast in a must, the original colony must increase in size until this critical population density of 30 million cells per fluid ounce is reached before fermentation can begin. The lag phase therefore represents a period of active yeast growth despite the apparent lack of activity at this time. Its duration is clearly dependent upon factors influencing yeast growth, e.g. temperature, availability of nutrients and oxygen, presence of sulphite and so on as well as the initial yeast concentration.
It follows from these remarks that, provided other conditions are favourable, inoculation of the must with an actively fermenting yeast starter should make for a short lag phase. Not only does this procedure ensure a fairly high initial yeast concentration but it also means that the yeast is already in an active state of growth and reproduction at the time of its introduction and does not have to be awakened from a period of dormancy, as would normally be the case if a culture were added directly to the must. The duration of the lag phase can usually be further decreased by ensuring that the optimum conditions for yeast growth and reproduction are established in the must. Thus, the effects of any sulphite should have been allowed to ameliorate by delaying inoculation with the starter for 24 hours, a plentiful supply of nutrients and oxygen should be available (the latter by stirring the must vigorously to promote aeration) and a temperature of 70°F.-80°F. should be maintained. Fermentation will then normally begin within a few hours, but even if it does take several days before this stage is reached no real harm will be done. It is, of course, advisable to avoid a lengthy lag phase whenever possible to minimise the risk of spoilage organisms developing and interfering with or even suppressing the growth of the yeast.
During the lag phase the yeast absorbs a great deal of the nutrients and dissolved oxygen from the must. It also oxidises a little of the sugar completely to carbon dioxide and water in order to obtain energy required for the rapid growth and reproduction which occurs at this time. Very little alcohol is produced under these circumstances, but because the amount of sugar lost in this way is relatively small, the loss of alcohol sustained as a result of this occurrence is also slight. The lag phase is thus a period of aerobic yeast respiration which lasts only for as long as oxygen either dissolved in the must or trapped in an air spaced above the must is available to the yeast. Once this supply of oxygen is exhausted, the yeast must obtain its metabolic energy solely from anaerobic fermentation. Should the oxygen supply fail before the yeast colony achieves a population density of 30 million cells per fluid ounce, active fermentation will be considerably delayed or may even fail to begin because yeast growth and reproduction become very slow under anaerobic conditions. It is therefore essential to provide the yeast with sufficient oxygen during the lag phase to permit rapid expansion of the colony. An unduly long lag phase may sometimes he observed simply because the yeast is becoming starved of oxygen. Aeration of the must by vigorous stirring may rectify matters in such cases, but the position with respect to nutrients, temperature, sulphite, etc., should also then be checked.
The end of the lag phase is usually marked by the appearance of tiny bubbles of carbon dioxide rising through the must and showing that active fermentation has commenced. The primary fermentation is considered to begin once carbon dioxide is being visibly evolved. Although at first only a gentle effervescence is observed, fermentation very quickly becomes extremely rapid and vigorous and large volumes of gas are given off. The reason for this sudden burst of activity is not difficult to determine. The primary fermentation, at least initially, is also a period of rapid yeast growth as well as the lag phase. The rate of expansion of the yeast colony during the lag phase reaches its maximum just before the primary fermentation begins. Since the yeast cells are then growing and reproducing very rapidly, they continue to do so during the initial stages of the primary fermentation aided by any residual oxygen which was not used up during the lag phase. As the onset of the primary fermentation automatically introduces anaerobic conditions, by forming a blanket of carbon dioxide over the now oxygen-free must, this extremely rapid growth of the yeast colony can only continue for a limited period of time. The yeast cannot maintain a fast growth rate with the energy obtained solely from anaerobic fermentation. Hence, the primary fermentation becomes progressively more vigorous only for a few days, the actual duration depending upon the availability of oxygen and nutrients, temperature and so on. Thereafter its vigor will moderate considerably within the next few days for reasons which will become clear shortly.
The overall picture can be seen even more clearly by studying the actual growth of the yeast colony itself. During the whole of the lag phase, the yeast population increases smoothly, the number of yeast cells doubling every few hours. This phase of activity continues unchanged into the initial primary fermentation. Up to this time, many more new cells are produced for each cell which dies, i.e. the birth rate considerably exceeds the death rate. As very much less energy is available for yeast growth and reproduction once anaerobic conditions have been established, the must is then unable to support this rapid population expansion any longer. Consequently, further yeast growth is severely curtailed. The duration of the vigorous primary fermentation is thus partly dependent upon how long a period elapses before all the available oxygen in the must becomes exhausted. Even when this stage is reached, however, fermentation continues for a few days with almost unabated vigour because at first the number of viable yeast cells remains fairly constant. The size of the colony will, of course, show no further marked increase during this time since yeast growth is now very restricted due to the onset of anaerobic conditions.
As a result, later in the primary fermentation the energy available for yeast growth and reproduction is insufficient to permit even the replacement of all the cells which die so that for a time the death rate exceeds the birth rate. After a few days, a balance is struck between the birth and death rates and the yeast population attains its optimum density commensurate with the prevailing conditions. It follows that the primary fermentation reaches a peak of activity in its early stages and subsequently becomes slower and more moderate according to the size of the yeast colony which the must can support.
The primary fermentation is also a period of rapid alcohol reduction, for large amounts of sugar must be fermented to maintain the vigor of the yeast colony. Hydrometer measurements made at this time will, of course, show this effect in the form of a very rapid gravity decrease. Little loss of alcohol is sustained as a result of the yeast oxidizing sugar completely to carbon dioxide and water during this period. Not only is the supply of oxygen essential for this purpose strictly limited but the metabolic rate of wine yeasts and to a slightly lesser extent that of baker’s yeast (but not that of poorly fermenting wild yeasts) is also so high that a good supply must be maintained before the alcohol yield is seriously depleted in this manner. The presence of ever-increasing quantities of alcohol in the must has other effects, however, for alcohol acts as an enzyme poison and slows down the metabolism of the yeast, thus decreasing I lie rate of fermentation. The increase in alcohol concentration and decrease in the size of the yeast colony observed during the latter is ages of the primary fermentation therefore both help to moderate its initial speed and vigor.
It is now clear that the rate and vigor of fermentation diminish fairly soon after primary fermentation begins, usually within about s 10 days, and subsequently a slower and more sedate fermentation is observed. This quieter and steadier phase of activity marks the beginning of the secondary fermentation. In view of the fact that this change is progressive rather than sudden in nature, however, the primary and secondary fermentation's merge imperceptibly together so that no sharp demarcation line can be drawn between I he two stages.
The rate of fermentation is slower during the secondary fermentation than in any previous stage. It may in fact become so slow that the only signs showing fermentation is proceeding at all are the presence of a ring of tiny bubbles round the perimeter of the must and a gradual but steady gravity decrease. Sugar is nevertheless slowly but surely being converted into alcohol and the amount of alcohol produced during this period approaches the theoretical yield more closely than at any other phase of fermentation. This behavior is typical of most strains of wine yeast and the slowness of the fermentation should be no cause for concern if periodic checks with the hydrometer show that a steady gravity decrease is occurring. On the other hand, if the gravity remains constant over an interval of 7-10 days then fermentation may have stuck for one of the reasons discussed later. In general, the winemaker should welcome a long slow fermentation since it has repeatedly been demonstrated that better quality wines are usually obtained under these conditions.
The secondary fermentation may continue for several months, but as time passes the rate of fermentation gradually decreases until finally it ceases completely and no further yeast activity is observed. Moreover, the accepted methods of stimulating fermentation, e.g. aeration, addition of nutrients, etc., become progressively less effective as the secondary fermentation proceeds towards completion. It follows that the alcohol produced during the fermentation must be exerting a stronger and stronger inhibitory action on the metabolism of the yeast as its concentration increases. Even- ually, so much alcohol has been formed that the enzyme system of the yeast becomes completely poisoned and fermentation consequently ceases. The rate of fermentation therefore gradually decreases with increasing alcohol production, and the alcohol concentration at which all yeast activity terminates is called the maximum tolerance of the yeast. Most wine yeasts have high alcohol tolerance and will normally produce 12%- 15% alcohol by volume with little difficulty, but certain strains, e.g. Madeira yeast, :an tolerate even higher alcohol concentrations. Indeed, up to 20%— 22% alcohol by volume has been claimed in some cases although more than about 18% alcohol by volume is rarely produced by natural fermentation. The increasing alcohol content luring this period also serves to kill off the weakest cells of the colony so that the death rate may slightly exceed the birth rate in the secondary fermentation, particularly in its later stages. This decrease in yeast population also, of course, makes for a slower rate of fermentation.
The rate of fermentation begins to decrease soon after the primary fermentation has commenced. Thereafter a progressive decrease in its rate is observed until finally fermentation ceases altogether because either the yeast has converted all the available sugar in the must into alcohol or it has reached its maximum alcohol tolerance. It has already been shown that this pattern of activity arises because the yeast population decreases and the alcohol concentration increases as fermentation proceeds towards completion.
In addition, it must be remembered that fermentation is essentially a series of chemical reactions. Thus, its rate will also be Impediment to some extent upon the temperature of the must for the simple reason that the rate of most chemical reactions approximately doubles with every 10°C. (18°F.) rise in temperature. Although this statement is also true for fermentation, it requires rather more careful interpretation in this case since living cells and heir enzymes provide the motivating power for its occurrence. Enzymes are delicate and rather unstable substances which are easily inactivated or denatured by heat so that the activity of all living cells shows a marked dependence upon temperature. Different organisms can tolerate different temperature levels and even the various strains of wine yeast show minor variation between themselves in this respect. In the case of most wine yeasts, however, their fermentation ability becomes seriously impaired if the cells are exposed to temperatures above 85°F.-90°F. for any length of time. Hence, any increase in the rate of fermentation which would be expected on purely chemical grounds is more than offset by the denaturation of the enzyme which occurs under these conditions. It follows that the rate of fermentation which would be expected on purely chemical grounds is more than offset by the denaturation of the enzymes which occurs under these conditions. It follows that the rate of fermentation reaches a maximum at a temperature slightly below that at which the enzymes in the yeast cells become inactivated. In the case of most wine yeasts, the maximum temperature at which fermentation should be conducted is around 75°F. - 80°F.
Conversely, lowering the temperature of the must would be expected to cause a decrease in the rate of fermentation, and this is indeed found to be the case. Musts held at low temperatures ferment very much more slowly than musts maintained at higher temperatures. Again, however, because fermentation occurs through the agency of living cells, it is found that yeast activity ceases if the temperature becomes too low. In this instance, the metabolism of the yeast has been so reduced by the low temperature that the cells cannot produce sufficient energy to support their continued growth. The yeast is then forced to hibernate until more favorable conditions are again established. Although the introduction of cross-bred hybrid yeasts now permits fermentation to continue at temperatures as low as 40°F.-45°F., the rate of fermentation even when these yeasts are employed becomes rather slow at temperatures below about 50°F.—55°F. simply because the enzymatic reactions responsible for fermentation proceed somewhat slowly under these conditions. The higher concentration of dissolved carbon dioxide attained at low temperatures also inhibits yeast activity slightly, but this factor alone is of minor importance.
It follows from this discussion that wine yeasts usually ferment most satisfactorily if the temperature of the must lies between about 55°F. and 80°F., and that a more rapid fermentation will occur the more closely the temperature approaches to about 80°F. The value of a long slow fermentation in connection with wine quality has already been able to conduct the fermentation at or near 80°F. Moreover, the alcohol tolerance of the yeast is somewhat reduced at higher temperatures. Since fermentation is an exothermic, i.e. heat- producing process, there is also the danger that a must held at 80°F. may reach too high a temperature because the excess heat produced during the very rapid fermentation occurring under these conditions cannot be lost quickly enough to the surroundings. Unless the must is cooled in some way to maintain its temperature below 80°F. - 85°F., fermentation may cease due to the yeast becoming inactivated by the heat. On the other hand, during the lag phase when active yeast growth and reproduction is taking place, it is clearly desirable to promote expansion of the yeast colony as quickly as possible in order to get the primary fermentation under way with the minimum of delay. Hence, a temperature of 75°F.—80°F. should be maintained during the lag phase (and luring the preparation of yeast starters for the same reasons). As soon as the primary fermentation begins, the temperature should be reduced to 60°F.-70°F. partly to prevent an unduly vigorous primary fermentation but mainly to ensure the long slow fermentation conducive to good quality is obtained.
When the fermentation is nearing completion, however, it is often advisable to raise the temperature to 75°F.-80°F. until all east activity finally ceases. This technique enables the yeast to utilize the maximum possible amount of sugar before it becomes exhausted and thus helps to stabilize the wine by reducing the chances of an inconvenient re-fermentation occurring later should le young wine be exposed to warmer temperatures at any time during storage. Sweet wines in particular should be treated in this lanner.
Almost all fermentation's will follow this pattern of activity as ley progress from inception to completion. The actual rate at each stage will depend upon the yeast population density, the alcohol concentration and the temperature. Marked deviations can nevertheless occur if other factors which influence yeast growth are unfavourable. For this reason, it is important to add an adequate supply of nutrients to the must prior to fermentation and to avoid too high an initial concentration of sulphite or sugar. Surprising as may seem, an excessive amount of sugar can prevent fermentation (the yeast cells are unable to withstand the osmotic pressure developed between the cell contents and the must and exhibit the phenomenon of plasmolysis). Fortunately, the must needs to contain about 4 lb. or more of sugar per gallon before this effect is observed and most winemakers would regard this quantity as excessive in any case.
At times fermentation will cease long before all the sugar in the must has been converted into alcohol. The fermentation is then said lo have stuck. This condition can easily be detected by means of the hydrometer, for a high gravity reading of 25 or above which shows no change over a week or ten days will be recorded. Fermentation can stick for a variety of reasons, all of which result in either the death of the colony or in its hibernation because the prevailing conditions are inimical to its continued normal existence. For example, too high or too low a temperature, an inadequate supply of nutrients, an excessively high concentration of sugar, oversulphiting and so on can all have this effect. Sometimes fermentation sticks because the yeast has reached its maximum alcohol tolerance without being able to deal with all the sugar in the must and a sickly oversweet wine results. At other times so much sugar may be present initially that fermentation hardly occurs at all and only a slightly alcoholic cordial is produced. Care must be taken to distinguish between these two extremes. A strong but over-sweet wine can and should always be used for blending and not treated as a stuck fermentation whereas a weakly alcoholic syrup is quite unsuitable for this purpose. In most cases, however, a stuck fermentation signifies that little alcohol has been formed and much unfermented sugar remains in the must. For this reason, stuck wines are very susceptible to bacterial infection and spoilage.
A stuck fermentation is obviously a highly undesirable occurrence which should be rectified as soon as possible. Unfortunately, any steps which are taken to remedy matters in the original must are unlikely to induce fermentation to restart even if the yeast colony is merely hibernating. A fresh yeast starter should therefore be introduced according to the following technique. The gravity of the must should first be reduced to about 80 or less with a similar sugar-free must or water if too high an initial concentration of sugar is responsible for sticking. In all cases, a fresh supply of nutrients should be added and the must stirred vigorously to promote aeration. An equal quantity of the must should next be mixed with an actively fermenting yeast starter and allowed to stand at a temperature of 75°F. - 80°F. until active fermentation is observed. This procedure must then be repeated, mixing an equal quantity of stuck wine and actively fermenting must each time, until the whole of the stuck wine is again fermenting. This rather laborious procedure is usually necessary when restarting a stuck fermentation. The new colony otherwise not infrequently fails to establish itself if it is added directly to the stuck must, even though an actively fermenting starter is employed, because the sudden change of environment may be too drastic under these rather unusual conditions.
