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3.1 What are yeasts?

A yeast is a unicellular fungus which reproduces asexually by budding or division, especially the genus Saccharomyces which is important in food fermentations (Walker, 1988). Yeasts and yeast-like fungi are widely distributed in nature. They are present in orchards and vineyards, in the air, the soil and the intestinal tract of animals. Like bacteria and moulds, they can have beneficial and non-beneficial effects in foods. Most yeasts are larger than most bacteria. The most well known examples of yeast fermentation are in the production of alcoholic drinks and the leavening of bread. For their participation in these two processes, yeasts are of major importance in the food industry.

Some yeasts are chromogenic and produce a variety of pigments, including green, yellow and black. Others are capable of synthesising essential B group vitamins.

Although there is a large diversity of yeasts and yeast-like fungi, (about 500 species), only a few are commonly associated with the production of fermented foods. They are all either ascomycetous yeasts or members of the genus Candida. Varieties of the Saccharomyces cervisiae genus are the most common yeasts in fermented foods and beverages based on fruit and vegetables. All strains of this genus ferment glucose and many ferment other plant derived carbohydrates such as sucrose, maltose and raffinose. In the tropics, Saccharomyces pombe is the dominant yeast in the production of traditional fermented beverages, especially those derived from maize and millet (Adams and Moss, 1995).

3.2 Conditions necessary for fermentation

Most yeasts require an abundance of oxygen for growth, therefore by controlling the supply of oxygen, their growth can be checked. In addition to oxygen, they require a basic substrate such as sugar. Some yeasts can ferment sugars to alcohol and carbon dioxide in the absence of air but require oxygen for growth. They produce ethyl alcohol and carbon dioxide from simple sugars such as glucose and fructose.

                                                 C₆H₁₂O_{6              Þ}              2C₂H₅OH               +              2CO₂

                                                 Glucose          yeast         ethyl alcohol              +              carbon dioxide

In conditions of excess oxygen (and in the presence of acetobacter) the alcohol can be oxidised to form acetic acid. This is undesirable if the end product is a fruit alcohol, but is a technique employed for the production of fruit vinegars (see later section on mixed fermentations).

Yeasts are active in a very broad temperature range - from 0 to 50° C, with an optimum temperature range of 20° to 30° C.

The optimum pH for most micro-organisms is near the neutral point (pH 7.0). Moulds and yeasts are usually acid tolerant and are therefore associated with the spoilage of acidic foods. Yeasts can grow in a pH range of 4 to 4.5 and moulds can grow from pH 2 to 8.5, but favour an acid pH (Mountney and Gould, 1988).

In terms of water requirements, yeasts are intermediate between bacteria and moulds. Bacteria have the highest demands for water, while moulds have the least need. Normal yeasts require a minimum water activity of 0.85 or a relative humidity of 88%.

Yeasts are fairly tolerant of high concentrations of sugar and grow well in solutions containing 40% sugar. At concentrations higher than this, only a certain group of yeasts – the osmophilic type – can survive. There are only a few yeasts that can tolerate sugar concentrations of 65-70% and these grow very slowly in these conditions (Board, 1983). Some yeasts – for example the Debaromyces - can tolerate high salt concentrations. Another group which can tolerate high salt concentrations and low water activity is Zygosaccharomyces rouxii, which is associated with fermentations in which salting is an integral part of the process.

3.3 Production of fruit alcohol

Alcohol and acids are two primary products of fermentation, both used to good effect in the preservation of foods. Several alcohol-fermented foods are preceded by an acid fermentation and in the presence of oxygen and acetobacter, alcohol can be fermented to produce acetic acid. Most food spoilage organisms cannot survive in either alcoholic or acidic environments. Therefore, the production of both these end products can prevent a food from undergoing spoilage and extend its shelf life.

Primitive wines and beers have been produced, with the aid of yeasts, for thousands of years, although it was not until about four hundred years ago that micro-organisms associated with the fermentation were observed and identified. It was not until the 1850’s that Louis Pasteur demonstrated unequivocally the involvement of yeasts in the production of wines and beers (Fleet, 1998). Since then, the knowledge of yeasts and the conditions necessary for fermentation of wine and beer has increased to the point where pure culture fermentations are now used to ensure consistent product quality. Originally, alcoholic fermentations would have been spontaneous events that resulted from the activity of micro-organisms naturally present. These non-scientific methods are still used today for the home preparation of many of the worlds traditional beers and wines.

Alcoholic drinks fall into two broad categories: wines and beers. Wines are made from the juice of fruits and beers from cereal grains. The principal carbohydrates in fruit juices are soluble sugars; the principal carbohydrate in grains is starch, an insoluble polysaccharide. The yeasts that bring about alcoholic fermentation can attack soluble sugars but do not produce starch-splitting enzymes. Wines can therefore be made by the direct fermentation of the raw material, while the production of beer requires the hydrolysis of starch to yield sugars fermentable by yeast, as a preliminary step (Stanier, Dourdoff and Adelberg, 1972).

Raw fruit juice is usually a strongly acidic solution, containing from 10 to 25 percent soluble sugars. Its acidity and high sugar concentration make it an unfavourable medium for the growth of bacteria but highly suitable for yeasts and moulds. Raw fruit juice naturally contains many yeasts, moulds, and bacteria, derived from the surface of the fruit. Normally the yeast used in alcoholic fermentation is a strain of the species Saccharomyces cerevisiae (Adams, 1985).

The fermentation may be allowed to proceed spontaneously, or can be "started" by inoculation with a must that has been previously successfully fermented by S. cerevisiae var. ellipsoideus. Many modern wineries eliminate the original microbial population of the must by pasteurisation or by treatment with sulphur dioxide. The must is then inoculated with a starter culture derived from a pure culture of a suitable strain of wine yeast. This procedure eliminates many of the uncertainties and difficulties of older methods. At the start of the fermentation, the must is aerated slightly to build up a large and vigorous yeast population; once fermentation sets in, the rapid production of carbon dioxide maintains anaerobic conditions, which prevent the growth of undesirable aerobic organisms, such as bacteria and moulds. The temperature of fermentation is usually from 25 to 30^oC, and the duration of the fermentation process may extend from a few days to two weeks. As soon as the desired degree of sugar disappearance and alcohol production has been attained, the microbiological phase of wine making is over. Thereafter, the quality and stability of the wine depend very largely on preventing further microbial activity, both during the "aging" in wooden casks and after bottling (Stanier et al, 1972).

At all stages during its manufacture, fruit juice alcohol is subject to spoilage by undesirable microorganisms. Pasteur, whose descriptions of the organisms responsible and recommendations for overcoming them are still valid today, first scientifically explored the problem of the "diseases" of wines. The most serious aerobic spoilage processes are brought about by film-forming yeasts and acetic acid bacteria, both of which grow at the expense of the alcohol, converting it to acetic acid or to carbon dioxide and water. The chief danger from these organisms arises when access of air is not carefully regulated during aging. Much more serious are the diseases caused by fermentative bacteria, particularly rod-shaped lactic acid bacteria, which utilise any residual sugar and impart a mousy taste to the wine. Such wines are known as turned wines. Since oxygen is unnecessary for the growth of lactic acid bacteria, wine spoilage of this kind can occur even after bottling. These risks of spoilage can be minimised by pasteurisation after bottling (Stanier et al, 1972).

Grape wine is perhaps the most common fruit juice alcohol. Because of the commercialisation of the product for industry, the process has received most research attention and is documented in detail.

The production of grape wine involves the following basic steps: crushing the grapes to extract the juice; alcoholic fermentation; maltolactic fermentation if desired; bulk storage and maturation of the wine in a cellar; clarification and packaging. Although the process is fairly simple, quality control demands that the fermentation is carried out under controlled conditions to ensure a high quality product.

The distinctive flavour of grape wine originates from the grapes as raw material and subsequent processing operations. The grapes contribute trace elements of many volatile substances (mainly terpenes) which give the final product the distinctive fruity character. In addition, they contribute non-volatile compounds (tartaric and malic acids) which impact on flavour and tannins which give bitterness and astringency. The latter are more prominent in red wines as the tannin components are located in the grape skins.

Although yeasts are the principal organisms involved, filamentous fungi, lactic acid bacteria, acetic acid bacteria and other bacterial groups all play a role in the production of alcoholic fruit products (Fleet, 1998).

Normal grapes harbour a diverse micro-flora, of which the principal yeasts (Saccharomyces cerevisiae) involved in desirable fermentation are in the minority. Lactic acid bacteria and acetic acid bacteria are also present. The proportions of each and total numbers present are dependent upon a number of external environmental factors including the temperature, humidity, stage of maturity, damage at harvest and application of fungicides. It is essential to ensure proliferation of the desired species at the expense of the non-desired ones. This is achieved through ensuring fermentation conditions are such to encourage Saccharomyces species.

The fermentation may be initiated using a starter culture of Saccharomyces cerevisiae – in which case the juice is inoculated with populations of yeast of 10⁶ to 10⁷ cfu/ml juice. This approach produces a wine of generally expected taste and quality. If the fermentation is allowed to proceed naturally, utilising the yeasts present on the surface of the fruits, the end result is less controllable, but produces wines with a range of flavour characteristics. It is likely that natural fermentations are practiced widely around the world, especially for home production of wine.

During alcoholic fermentation, yeasts are the prominent species. The composition of fruit juice – its acid and sugar level and low pH favour the growth of yeasts and production of ethanol that restricts the growth of bacteria and fungi.

In natural fermentations, there is a progressive pattern of yeast growth. Several species of yeast, including Kloeckera, Hanseniaspora, Candida and Metschnikowia, are active for the first two to three days of fermentation. The build up of end products (ethanol) is toxic to these yeasts and they die off, leaving Saccharomyces cerevisiae to continue the fermentation to the end. S. cerevisiae can tolerate much higher levels of ethanol (up to 15% v/v or more) than the other species who only tolerate up to 5 or 8% alcohol (Fleet, 1998). Because of its tolerance of alcohol, S. cerevisiae dominates wine fermentation and is the species that has been commercialised for starter cultures.

Traditionally, fermentation was carried out in large wooden barrels or concrete tanks. Modern wineries now use stainless steel tanks as these are more hygienic and provide better temperature control. White wines are fermented at 10 to 18º C for about seven to fourteen days. The low temperature and slow fermentation favours the retention of volatile compounds. Red wines are fermented at 20 to 30ºC for about seven days. This higher temperature is necessary to extract the pigment from the grape skins (Fleet, 1998).

There are several variables which can affect the fermentation process and final quality of wine. Factors which are most important to control are:

• the clarification and pre-treatment of juice
• chemical composition of the juice
• temperature of the fermentation
• the influences of other micro-organisms.

Excessive clarification removes many of the natural yeasts and flora. This is beneficial if a tightly controlled induced fermentation is desired, but less so if the fermentation is a natural one. Long periods of settling out however, encourage the growth of natural flora, which can contribute to the fermentation.

The main consituents of grape juice are glucose (75 to 150 g/l), fructose (75 to 150 g/l), tartaric acid (2 to 10 g/l), malic acid (1 to 8 g/l) and free amino acids (0.2 to 2.5 g/l). The main reaction is the fermentation of glucose and fructose to ethanol and carbon dioxide. However, the presence of nitrogenous and sulphurous products also contributes to the fermentation. The addition of sulphur dioxide to the juice delays the growth of yeast, but does not necessarily inhibit growth of the non-Saccharomyces strains. Fruits generally contain sufficient substrates - soluble sugars - for the yeast to ferment and convert into an acceptable concentration of alcohol. Sugar can be added to fruit juices with a low sugar content, to increase the amount of fermentable substrate.

Temperature has an impact on the growth and activity of different strains of yeast. At temperatures of 10 to 15° C, the non-Saccharomyces species have an increased tolerance to alcohol and therefore have the potential to contribute to the fermentation.

Other micro-organisms have the potential to influence wine production at all stages of the process. Prior to harvest, yeasts grow on the surface of grapes. Fungicides are used in an attempt to control their growth, but these disturb the natural balance of flora, thus making it difficult to carry out a ‘natural’ fermentation. Overuse of fungicides can lead to the development of resistant strains of yeast which have the potential to produce toxins which destroy the desirable yeast species. These yeasts are known as ‘killer’ strains. Other microbes have further chances to influence the fermentation during the clarification process, after fermentation and during maturation and bottling when acetobacter species can oxidise the alcohol and produce acetic acid.

About two to three weeks after the alcoholic fermentation is finished wines often undergo a malo-lactic fermentation. This occurs naturally and lasts for about four weeks. It is a lactic acid fermentation, initiated by lactic acid bacteria resident in the wine. Inoculating the fermented wine with cultures of Leuconostoc oenos can start the process if it is desired. The main reaction of these bacteria is the decarboxylation of L-malic acid to L-lactic acid, which decreases the acidity of the wine and increases its pH by about 0.3 to 0.5 units. Wines produced from grapes grown in colder climates tend to have a higher concentration of malic acid and a lower pH (3.0 to 3.5) and the taste benefits from this slight decrease in acidity. The benefits of this process are that it imparts a more mellow flavour to the wine. The growth of malo-lactic bacteria also contributes to the taste of the wine. Wines that have undergone a malo-lactic fermentation appear to be less susceptible to any further damage from other bacteria. This could be because L. oenos has used up all available substrate, or it may have secreted bacteriocins which prevent the growth of other species (Fleet, 1998). Although the malo-lactic fermentation seems to be a useful process, not all wines benefit from it. Wines produced from grapes in warmer climates tend to be less acidic (pH > 3.5) and a further reduction in acidity may have adverse effects on the quality of the wine. Decreasing the acidity also increases the pH to values which can allow spoilage organisms to multiply. It is difficult to prevent the malo-lactic fermentation from taking place naturally, especially later on after the wine has been bottled. In low acid wines, the acidity may be adjusted after this fermentation has taken place. The malo-lactic fermentation can be prevented by controlling several factors: the wine pH (< 3.2); ethanol content (> 14%) and levels of sulphur dioxide (>50 mg/l). The bacteriocin nisin can also be used to control the growth of malo-lactic bacteria. However the subtle blend of aromas and flavours that contribute to the final taste may be lost by such stringent control.

The conversion of malic acid to lactic acid is one of the main reactions carried out by wine lactic acid bacteria. L oenos needs to be present in significant numbers (greater than 10⁶ cfu/ml) for the reaction to take place at a suitable pace. The bacteria use residual pentose and hexose sugars in the wine as a substrate for growth. The main reaction is the deacidification (or decarboxylation) of malic acid. In addition to this, the by-products of the reaction impart flavours and aromas to the wine.

During storage, wines are prone to non-desirable microbial changes. Yeasts, lactic acid bacteria, acetic acid bacteria and fungi can all spoil or taint wines after the fermentation process is completed. The changes that occur are increased acidification through the formation of acetic and other acids from alcohol; increased carbonation through a secondary fermentation of residual sugars and flavour changes through the metabolism of numerous compounds.

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