THE BASIC MORPHOLOGY OF WINE YEASTS

Saccharomyces Cerevisiae cells observed under the microscope

AND THEIR ROLE IN FERMENTATION

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Contents

Yeasts 2. 3. 4. Naming conventions The process of alcoholic fermentation The yeast fermentation equation

5. Reproduction and Morphology Budding Spores Morphology 6. 7. Using yeasts in winemaking Commercial yeasts

ed fermentations 8. Indigenous yeasts Spoilage yeasts Practical aspects of indigenous fermentations Conditions for development of yeasts

9.

of fermentation Limit of fermentation and temperature Critical temperatures for yeast Influence of aeration Nutritional needs of yeasts 10. References

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1. YEASTS Yeasts are unicellular, microscopic fungi that reproduce by budding and binary fission. They decompose sugar into alcohol and carbon dioxide. The size of yeast cells varies according to species. Diameters can range from 2 to 10 m. 1 m = 10
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meters.

In general, cells find the energy they need in two ways, both of which involve the dissipation of organic matter. Breathing. Breathing takes oxygen from the air, produces advanced molecular fission, and releases a lot of energy. Fermentation. Fermentation takes place in the absence of oxygen. It is inefficient because the dissipation of matter that it promotes is incomplete. Yeasts have to transform a great deal of sugar into alcohol to satisfy their energy needs.

2. NAMING CONVENTIONS There exists a considerable number of yeast species differentiated by their shape, their properties, their mode of reproduction, and by the way they transform sugar to alcohol. Wine yeasts belong to a dozen genera each divided into species. They are designated by a double name in Latin, the first corresponding to the genus and the second to the species. For example, the yeast named Saccharomyces ellipsoideus belongs to the genus Saccharomyces (literally the sugar fungus, which transforms sugar) and the species ellipsoideus (elliptical in shape). In modern winemaking, the elliptic shaped Saccharomyces cerevisiae is the preferred yeast species. Within the Saccharomyces cerevisiae species, there are many strains that show very minor genetic differences. Winemakers may prefer a specific commercial strain of yeast for its known attributes over another strain. 3. THE PROCESS OF ALCOHOLIC FERMENTATION

Fermentation of crushed grapes and juice is a common occurrence and has been happening naturally and spontaneously for thousands of years. Damaged or split berries release their juice and indigenous yeast present on the grape skin will begin to use the juice as a food source. Spontaneous fermentation will commence. Today, winemakers typically use commercially selected strains of yeast, which they add to juice and must (juice, skins and seeds), as well as indigenous yeasts. Once fermentation is underway, sugar is transformed into alcohol and carbon dioxide gas. The must goes cloudy and gets hot. The temperature may rise to as much as 35C unless refrigeration is used. Bubbles of carbon dioxide gas
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rise causing the must to seethe. As the sugar is fermented, the liquid loses its sweet taste and becomes vinous. Wine is made. The chemical mechanism of sugar fermentation is extremely complex. The most important transformations comprise no less than 30 or so successive reactions, bringing into play a great many enzymes. The enzymes are the tools of the yeasts adapted to one stage of the transformation. Each reaction necessitates a different tool, a different enzyme. The by-products that have already been mentioned are like the remnants of these multiple reactions. The by-products are important to the final product, the finished wine.

Yeasts initially, preferentially use the glucose portion of the grape sugars, building up cell biomass with little alcohol produced. As fermentation proceeds, ethanol is produced by utilization of both the glucose and fructose portion of the grape sugars. A small percentage of the fructose portion of the grape sugars is usually consumed last. This occurs because yeasts are required to provide active transport through the cell membrane for fructose whereas yeasts do not have the same requirement for glucose. Fructose is twice as sweet as glucose. 4. THE YEAST FERMENTATION EQUATION The following is a simple but for our purposes, perfectly adequate, balanced chemical equation to represent the fermentation process. C6H12O6 grape sugars primarily glucose and fructose 180 grams yeast 2C2H5OH ethyl alcohol (ethanol) 92 grams + 2CO2 carbon dioxide gas 88 grams + heat

The weights given are based on molecular weights of the products and reactants. In fact the amounts of alcohol and carbon dioxide yielded by the fermentation process are somewhat less than shown, say 90-92%, because there are some other fermentation products produced. Some other fermentation products include Glycerol can contribute to smooth mouthfeel, viscosity and some sweetness Succinic acid. Its bitter-salty taste limits it use in wine acidification Butylene glycol (sweet tasting, Austrians know all about this)
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Acetic acid, CH3COOH, around 0.5 g/L can be produced during fermentation. Higher amounts may be formed by bacterial or oxidative yeast activity during and after fermentation. Ethyl acetate (also called acetone) is produced concurrently with acetic acid, usually in the ratio 1 part ethyl acetate to 5 parts acetic acid. Ethyl acetate is more readily detected organoleptically (aeroplane glue, nail varnish remover) than is acetic acid (vinegary). Ethyl Acetic acid bacteria can directly synthesize ethyl acetate. As well ethyl acetate can be formed by acetic acid reacting with ethanol. Lactic acid (yeast will degrade a small amount of naturally occurring malic acid during fermentation, especially Schizosaccharomyces pombe, which may degrade all the malic acid but also produces other off-characters) Higher alcohols (fusel oils) tend to be hot and medicinal Acetaldehyde, CH3CHO, is the major aldehyde found in wine. Aldehydes are carbonyl compounds distinguished by the terminal location of the carbonyl (C=O) functional group on the molecule. Combined with other oxidized compounds it contributes to the fragrance of sherry and other oxidized wines. Acetaldehyde is one of the early metabolic by-products of fermentation. As fermentation approaches completion, acetaldehyde is transported back into yeast cells and reduced to ethanol. Pyruvic acid Acetoin (buttery taste produced by yeast) And other compounds

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5. REPRODUCTION AND MORPHOLOGY Yeasts are found in three classes of fungi, characterized by their reproduction mode: Sac fungi (Ascomycetes) Club fungi (Basidiomycetes) Imperfect fungi (Deuteromycetes) The yeasts found on the surface of the grape and in wine belong to Ascomycetes (Capable of reproducing sexually as well as by budding. Important in fermentation) Deuteromycetes (Only able to reproduce by budding. Some species of Candida are important as spoilage yeasts.) The majority of wine yeasts show two possible methods of reproduction according to prevailing conditions. 1. Vegetative or asexual reproduction by budding Bi-polar e.g. Kloeckera apiculata, an indigenous, spoilage yeast that extrudes daughter cells at opposite poles of their cell creating a lemon shape over successive generations. Multilateral e.g. Saccharomyces cerevisiae, a yeast that produces buds (daughter cells) from any part of the external surface of the cell. Bud fission e.g. Schizosaccharomyces pombe (a mixture of budding and binary fission). Sexual reproduction by the formation of ascospores, which after germination give rise to more yeasts.

2.

Budding As soon as a yeast cell is added to grape juice or must, a swelling can soon be seen appearing on its circumference that gradually gets bigger at the same time that the shape of the small daughter cell begins to be defined. When the two cells are approximately the same size, they separate and budding will continue involving both the new daughter cell and the original mother cell. Under ideal conditions, only two hours are needed to double the yeast population with up to six hours needed as yeast nutrients become limiting. Spores When sporiferous yeast diploid cells are in a hostile nutritive medium (for example, depleted of fermenting sugar, poor in nitrogen, and very aerated) they stop multiplying. Some transform into a kind of sac with a thick cell wall. These sacs are called asci. (Refer to figures 5 and 6). Asci are robust and are able to survive under harsh conditions, for example dehydration, contact with chemical agents such as sulphur dioxide, and heat, that normally would be fatal to yeasts. Each asci contains four haploid ascospores issued from meiotic division of the nucleus. Grape must and wine are not propitious to yeast sporulation. Wine yeasts, both indigenous and selected, do not sporulate easily and when they do they often produce non-viable cells. In favorable conditions, i.e. nutritive sugar-enriched media, the haploid ascospores germinate, breaking the cell wall of the ascus, and begin to multiply. In S. cerevisiae, the ascospores have two mating types. Sexual coupling occurs between two cells of different mating type. The vegetative diploid yeast cell resulting from cellular and nuclear fusion of the ascospores multiplies by budding. Morphology
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Yeasts found in vinification will typically present one of the four following shapes: elliptical or ovoid, elongated (in the shape of a sausage), spherical, and apiculate. In modern winemaking, where inoculations of known yeast strains are preferred, the yeast cell shape viewed under the microscope will commonly be elliptical, the shape of Saccharomyces cerevisiae cells.

Figure 1. Various shapes of wine yeasts. From left to right: Elliptic yeast of the genus Saccharomyces Sporulated elliptic yeast Round yeast, previously known as Torula Tiny elongated yeast, Torulopsis stellata Apiculate yeast, Hanseniaspora Large apiculate yeast, Saccharomycodes ludwigii (Peynaud, 1984)

Figure 2. Observation of two enological yeasts species having an apiculated form a) Hanseniaspora uvarum b) Saccharomycodes ludwigii (Ribreau-Gayon et al, 2000)

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Figure 3. Binary fission Schizosaccharomyces. Formation of a cell wall occurs between mother and daughter followed by separation. (Ribreau-Gayon et al, 2000)

Figure 4. Multilateral budding characteristic of Saccharomyces spp. occurs on the shoulder area of the yeast. Each bud arises at a location separate from others. Upon separation of daughter cells, the mother is left with bud scars. Each budding cycle depletes the mother cells membrane by approximately one half. Cell membrane synthesis occurs under aerobic conditions. Thus, after three or four budding cycles, the fermentation must complete with a stationary-phase population. Older cells of multilateral budding Brettanomyces and Dekkera spp. may exhibit a cell shape suggestive of restrictive polar budding. Cells are appear boat-shaped and described as ogival. (Fugelsang, 1996)

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Figure 5. Stages of yeast development. From left to right: budding vegetative cells, ascospore development, spore release from ascus. (Jackson, 2000)

Figure 6. Saccharomyces cerevisiae cells placed on a sugar-agar medium for several weeks. Asci containing ascospores can be observed. (Ribreau-Gayon et al, 2000)

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6. USING YEASTS IN WINEMAKING Fermentation of grape juice and must is initiated by either inoculating the juice or must with a commercial yeast, or by allowing a spontaneous fermentation to occur using indigenous yeasts present on the grape skins.
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Yeast populations in fully fermenting grape juice and must are extremely dense, in the region of 80 million to 120 million, i.e. around 10 yeasts per milliliter. In one drop of fermenting grape juice there may be 5 million yeasts. Yeast populations will need to be this high if the fermentation is to go to complete dryness, i.e. if most of the grape sugars are to be completely fermented. 7. COMMERCIAL YEASTS Most commercial yeasts are strains of Saccharomyces cerevisiae that have been developed and selected for particular characteristics. Winemakers will choose one commercial strain over another because of its known attributes. It is common practice in New World winemaking countries such as New Zealand, Australia, USA and Canada, to inoculate grape juice or must with commercial yeasts in an attempt to ensure consistency of wine style known performance attributes protection of varietal characters greater control of fermentation Commercial yeasts are sold as dried powders and several American and European brands have been on the market for up to forty years. They are prepared by vacuum dehydrating yeasts in hot air with a protective support present. They are not freeze-dried preparations. Yeasts are easily kept refrigerated as dried powders, and are available all year round. They can be used in sparkling wine production, fermentation of stored musts or juices, and to re-inoculate stuck ferments (where a fermentation has stopped prematurely with unwanted residual sugar still remaining). Desirable characteristics of wine yeasts include 1. 2. 3. 4. 5. 6. The efficient conversion of grape sugar to alcohol The ability to effect a quick initiation of fermentation, say within 48 hours Sulphur dioxide tolerance The ability to carry out even fermentation The ability to ferment at low temperatures, important if you want to retain fruity characters The ability to ferment to dryness which requires that they are alcohol tolerant and can ferment in the presence of relatively high alcohol concentration 7. Good flocculation ability following fermentation to aid clarification 8. The production of a desirable fermentation bouquet 9. Low foaming 10. Low H2S or mercaptan formation 11. Relatively high glycerol production to aid the sensory qualities of the wine 12. Relatively low alcohol production to avoid hotness 13. Low acetic acid formation

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To be really efficient, to would be better if indigenous yeasts on the grapes and in the winery, were eliminated before juice or must was inoculated with commercial yeasts. Theoretically at least, it is possible that indigenous yeasts, better adapted, could outclass the yeasts introduced. However it is not possible to sterilize vintage. Elimination or containment of indigenous yeasts is more easily achieved in white wine making, where the must is homogeneous and allows for better mixing, than in red winemaking Inoculation using commercial yeasts is particularly recommended if fermentation is slow to start. It can be useful in cold years when grapes arrive at the winery at a temperature below 15oC. Commercial yeasts may better handle fungicide residues. They are generally more tolerant of sulphur. Inoculation will be required if white musts are drastically sedimented, especially when clarified by centrifugation or some other means. With inoculation, defective wild microflora, such as yeasts producing ethyl acetate, hydrogen sulfide, or sulfur dioxide, or having other undesirable characteristics, can be supplanted. Practical aspects of inoculated fermentations Inoculation is carried out with the juice at 15C or higher. Keeping the juice warm for say 24 hours after inoculation may be helpful to ensure a good start to fermentation. Yeast and supplements are prepared and added to juice or must as follows. Yeast is typically added at approximately 0.2g/L for whites and 0.3g/L for reds. Lower amounts might be used if the yeast strain was very vigorous. More could be used if the strain was less vigorous. Generally yeast is re-hydrated in warm water at 40C. It is sprinkled (without stirring) onto a volume of water, equal (in millilitres) to 10 times the weight of yeast (in grams). After 20 minutes or so, a small amount of juice may be added to the re-hydrating yeast to help acclimatise the yeast to the sugar levels that will be encountered in the juice or must, and to reduce the temperature. Ideally the re-hydrated yeast should be added to the juice or must when it has pretty much reached the same temperature of the juice or must it is being added to. DAP or di-ammonium phosphate is a nitrogen supplement for yeasts. It is mixed up in warm water or juice and added to the juice or must. DAP is added at a rate of around 0.4g/L. However it is preferable not to add all in one hit. Typically we would add 0.2 g/L at the time of yeast inoculation, 0.1 g/L day later and a further 0.1 g/L a day after that. This is to ensure that yeasts also take up amino acids. Yeasts can use nitrogen in the form of ammonia, or the ammonium ion, which are supplied by DAP and which are also contained in grapes. Yeasts also need amino acids. The amino N content in a grape must is typically 2 3 times higher than ammonia N. If a large amount of DAP is initially added, this balance is shifted and can cause metabolic disturbances in yeast cells, H2S production. Amino acids will not be taken up in a pattern that is beneficial to the health of the fermentation. Yeasts need their amino acids. Amino acids are also important flavour and aroma pre-cursors in a wines sensory profile. Dont let yeasts spoil their dinner by gorging on ammonia first. Superfood is a proprietary commercial product sold as a complete yeast nutrient blend. It is usually added at a rate of between 0.1 g/L and 0.2 g/L at the time of yeast inoculation. Superfood comes in the form of a powder and is mixed into a slurry with water or juice before being added to the juice or must. Superfood contains yeast hulls, yeast extract, DAP (around 33%), minerals and vitamins. Yeast hulls (yeast cell walls, cell membranes, envelopes cellularies) are yeast cell membranes left behind when yeast extract is prepared. They help yeasts protect their cell membranes against alcohol toxicity and heat and cold shock. Yeast hulls absorb pesticide residues, toxic short chain fatty acids produced by yeasts, and other inhibitory compounds. Yeast hulls are normally added at a rate of between 0.1g/L and 0.25 g/L at the time of yeast inoculation. Yeast hulls come in the form of a powder and are mixed into a slurry with water or juice before being added to the juice or must.
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8. INDIGENOUS YEASTS Rather than inoculate a juice or must with commercial yeasts, a winemaker may prefer to rely on indigenous yeasts present in the juice or must and allow fermentation to occur spontaneously with a view to obtaining increased complexity from a succession of different yeasts involved. Indigenous yeasts refer to yeasts that occur naturally in the vineyard or in the winery. In the vineyard indigenous yeasts are found on ripe grapes from veraison onwards, particularly around the time of harvest. They are brought into winery, onto presses, tanks and other equipment, with the grapes and develop and proliferate there with great speed. Saccharomyces species are particularly important in un-inoculated fermentations. Yeasts overwinter in the soil. Few exist on green grapes. In the vineyard in summer, midges and other insects pick up and spread yeasts and other organisms. As well as good yeasts, a number of other microorganisms are found on grapes including mycodermic yeasts or flower ferment, molds, lactic acid bacteria, and acetic acid bacteria. The yeasts most commonly found in combination in musts and juices are Saccharomyces cerevisiae, the common elliptical yeast, Kloeckera apiculata, the tiny apiculate yeast and Hanseniaspora uvarum, also apiculate These three species alone represent at least 90% of the strains responsible for fermentation. Principal species found in vinification Brettanomyces intermedius Candida sake Hanseniaspora uvarum Hansenula Kloeckera apiculata Kluyveromyces veronae Pichia membranaefaciens Saccharomyces aceti, bailii, bayanus, cerevisiae, oviformis Schizosaccharomyces pombe Torulopsis stellata Spoilage Yeasts A number of indigenous yeasts are regarded as spoilage yeasts and are undesirable in winemaking. They produce off-flavors and taints and other problems. They contaminate containers and equipment. They are typically resistant to alcohol, sulfur dioxide, and to anaerobic conditions, and can remain alive in wine in a latent state for months. The presence of these yeasts in stored wine can affect the clarity and result in the formation of deposits. When the wines contain reducing sugars, a genuine fermentation may be set off and the wine will go gaseous. These disturbances can occur not only during tank-life, but in the barrel, cask or bottle too. Spoilage yeast, not really a fermentation yeast, can contaminate barrels Spoilage yeast Spoilage yeast Spoilage yeast Spoilage yeast S. bayanus is of interest S. cerevisiae produces clean ferments with desirable characters and low volatile acidity S. oviformis has high-alcohol generating powers Able to degrade malic acid, de-acidifying the must, but can also produce off-characters Specific to grapes attacked by noble rot

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Practical aspects of indigenous fermentations Grape skins host very few Saccharomyces yeasts but many non-Saccharomyces yeast species. The majority are Kloeckera apiculata (and related Hanseniaspora uvarum), as well as other species. Most un-inoculated ferments, and even many inoculated ones in which the added yeasts die because of poor viability or mishandling, begin with non-Saccharomyces vineyard yeasts. Kloeckera tolerates at least 70 mg/L SO2, and is naturally cold tolerant. Saccharomyces is not. Reds soaking at less than 10C, or white juice warming up slowly after racking are prime candidates for Kloeckera growth. Kloeckera can make ethyl acetate (smells like nail polish remover) just before fermentation, but if a strong, clean fermentation ensues quickly there may be little harm done. If there is bumpy, slimy scum, or stringy floating clumps, it is more serious. Within hours, Kloeckera depletes the must of vitamins, particularly thiamine, leaving Saccharomyces without this important vitamin unless more is added at the right time. Kloeckera also produces acetic acid, which can act synergistically with alcohol to cause stuck ferments. Yeast supplements can be added when Saccharomyces can be seen under a microscope, and not before. Alternately some nutrients can be added after an initial 1 - 3Brix drop and the rest of the nutrients in portions until mid-fermentation. (PROS, 2004) In general we would not add sulphur if we wished to encourage an indigenous ferment. We would include some light lees or solids in a white ferment. We would quickly heat juice up, after cold settling, to encourage an indigenous fermentation. Kloeckera will kick in at lower temperatures and fall over around 3% alcohol. We dont want this particular yeast species to become too dominant, as among other things it tends to produce high VA. Notwithstanding it is one of a number of species that can contribute positively to complexity. Saccharomyces tend to kick in around 12C. Thus it is helpful if the must is quickly heated to this temperature so Kloeckera has a limited influence. Also oxygenate for a couple of hours to encourage indigenous fermentation.

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9. CONDITIONS FOR DEVELOPMENT OF YEASTS Yeasts have their precise needs in terms of nutrition and environment. They are very sensitive to temperature. They need oxygen. They need to be appropriately fed with sugars, mineral elements, nitrogenous substances, and vitamins. Temperature Yeasts only develop properly in the temperature range 15 - 35C. Below 13 or 14oC, the start of fermentation is practically impossible or else it is so slow that there is a danger of a spontaneous indigenous fermentation starting (usually Kloeckera apiculata). Fermentation cannot be conducted properly over 35oC. If the temperature is reached gradually, the yeasts stop working and die, sometimes at temperatures as low as 30 or 32oC. On the other hand, exceptional circumstances have been seen where the fermentation of an entire vintage, warmed throughout, started off at very high temperature (40 45oC), with the help of special yeasts, then finished very quickly as it cooled down. Above these temperatures and in wine in the presence of alcohol, yeasts are killed in a few minutes. This is sterilization by heating, or pasteurization. Temperature and the speed of fermentation Sugar transformation speeds up with a rise in temperature (at least up to a point). Fermentation is much faster at 30oC than at 25oC and at 25oC than at 20oC and its speed doubles for each 10oC variation. For each extra degree (centigrade) of temperature, yeasts transform 10% more sugar in the same period of time. Above 35oC, if at first fermentation is faster, it stops earlier by a kind of wearing out of the yeasts. Limit of fermentation and temperature The amount of sugar yeasts can transform, or the alcoholic strength they can attain, depends on the temperature. The higher the temperature, the quicker fermentation starts, but the sooner it stops and the lower the alcoholic strength attained (due to volatilization of alcohol). The maximum yeast population is lower at higher temperatures. When a high alcoholic strength is desired, the fermentation temperature must be kept fairly low. Everything happens as if the yeasts get tired all the more quickly the faster they work, at higher temperatures. In these conditions, they do not withstand alcohol easily, they are less able to assimilate the nitrogenous substances, they lose sterols more quickly, their reproduction falters and the fermentation comes to a halt. Critical temperatures for yeast The ideal temperature for making red wine is between 26 and 30oC. It is a compromise between the need to have a sufficiently rapid fermentation with thorough maceration and at the same time avoid fermentation stoppage through overheating. For making white and rose wines, the temperature to be preferred is lower, around 18 to 20oC. The notion of critical temperatures for fermentation is an important factor to consider in winemaking. Critical temperature is the temperature above which yeasts no longer reproduce, and die, making fermentation slow down, then stop. It is difficult to define a precise limit and better therefore to speak of a risk zone. This may vary in fact according to the aeration, richness of the must, nutritive factors for the yeasts, and the actual nature of these. In temperate regions the critical temperature is generally set above 30 - 32oC and in hot regions a little beyond this.
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The winemaker should not wait for the temperature in the tank to reach the risk zone before thinking about cooling. Cooling ought to intervene well before. What is important is to avoid crossing into this zone. The winemaker should avoid reaching this risk zone in the first place and maintain a favorable temperature, below critical temperature, to avoid destroying the yeasts. Influence of aeration Yeasts need oxygen to multiply. In a total absence of air, they only create a few generations and their growth stops. Then all it needs is to give them a little air again for budding to restart. If the state of asphyxia is prolonged, the majority of cells die. One yeast cell deprived of oxygen, finds the energy it needs in the transformation of sugar. But to get prolonged fermentation and obtain fermented products rich in alcohol, new generations of yeasts must constantly be forming and traces of oxygen become indispensable. This need for oxygen is, in a way, indirect. Yeasts require oxygen to synthesize the sterols and assimilate the fatty acids, which they need for cell growth. The sterols are the organic substances with several cycles of carbon atoms and with an alcohol function. They are the source of several hormones and vitamins whose biological importance is considerable. At the beginning of fermentation, the first generations of yeasts benefit from the reserves of sterols from the mother cells, then sterols from the natural environment. If fermentation is carried on in a shortage of air, the sterols are used up and are not renewed. Oxygen is indispensable to sterol synthesis and thus to the continuation of fermentation. All steps in which grapes are handled (crushing, de-stemming, pumping through, or again, for white grapes, draining off and pressing) make sure of a first aeration, helpful in getting fermentation started. Following this, the more oxygen the yeasts manage to get, the faster the yeasts multiply.

Nutritional needs of yeasts Yeasts have absolute requirements for certain nutrients in must and juice where they develop. Their needs in terms of sugar and mineral substances are easily satisfied. Their needs in respect of assimilable nitrogenous matter are less easily satisfied. Yeasts consist of 25-60% nitrogenous substances. To form their cells and reproduce, they need to find in their environment sufficient nitrogen in a form easy to use. Nitrogen in the form of ammonia or the ammonium cation, NH4 is the primary nitrogenous food consumed by elliptic yeasts, followed by certain free amino acids, such as glutamic acid. In 36 hours of fermentation, yeasts drain the must of its assimilable nitrogen and of many other nutritive elements. The rest of the fermentation goes on with starved yeasts in a state of deficiency. Toward the end of fermentation, they give the wine its amino acids back by excretion. In certain years, juice and must will be naturally low in YAN (yeast assimilable nitrogen. This may be exacerbated by soil conditions, very ripe grapes, or by fruit being infected with botrytis. DAP can be used to help supplement the supply of ammoniacal nitrogen, however, EC regulations restrict the use of DAP to a maximum of 0.3 g/L.
+

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10. REFERENCES Boulton, R.B., Singleton, V.L., Bisson, L.F., Kunkee, R.E. (1995). Principles and Practices of Winemaking. New York: Chapman and Hall. Fugelsang, K.C. (1996). Wine Microbiology. New York: Chapman and Hall Jackson, R.S. (2000). Wine Science: Principles, Practice Perception. London: Academic Press Nelson Marlborough Institute of Technology. (2004). Science One Laboratory Manual. Blenheim: NMIT. Pacific Rim Oenolgy Services. (2004). Harvest 2004 Products and Services. Marlborough, NZ: PROS Peynaud, E. (1984). Knowing and Making Wine. New York: Wiley-Interscience, Phaff, H.J., Miller, M.W., Mrak, E.M. (1978). The Life of Yeasts: Their Nature, Activity, Ecology and Relation to Mankind. Cambridge, Massachusetts: Harvard University Press Ribreau-Gayon, P., Dubourdieu, D., Donche, B., Lonvaud, A. (2000). Handbook of Enology Volume 1, The Microbiology of Wine and Vinifications. London: John Wiley & Sons Ltd

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