Foodborne Microorganism – Yeasts

The term “yeast”, which is similar to mold, is often used, but it can be difficult to define. It refers to fungi that are not filamentous, but unicellular, ovoid, or spheroid, and can reproduce by budding and fission. Yeasts can be beneficial or harmful to foods. Yeast fermentations play a role in the production of bread, wine, vinegar, and surface-ripened dairy products. Yeasts can be grown for enzymes or for food. When yeasts cause spoilage in sauerkraut, fruit juices and sirups as well as honey, jellies, meats and wine, they are considered undesirable.

General Characteristics of Yeasts

Although yeasts are classified primarily on the basis of their morphological characteristics and their physiological properties, Ones are more important for the food microbiologist.

Morphological Characteristics

Microscopic examination is used to determine the morphological characteristics and physiology of yeasts.

Form and Structure 

• You can find yeasts in a variety of shapes, including spherical, ovoid, pear-shaped, cylindrical, triangular or even elongated to make a true or false mycelium.
• They can also vary in their size. The cell wall is visible and may be either metachromatic or albuminous.
• To show the nucleus, special staining is required.

Reproduction

• Most yeasts reproduce asexually through multilateral or polar budding. This is when some of the protoplasms bulges from the cell wall. The bulge then grows in size until it eventually walls off and becomes a new yeast cell.
• Some yeasts, particularly those of the film yeasts have a tendency to produce bud from a tube-like projection from their mother cells.
• Replicated nuclear material can be divided between mother and daughter cells.
• Few yeast species reproduce by fission. One other reproduces through a combination fission/budding.
• Ascomycotina is a “true” yeast that reproduces sexually.
• Ascospore formation occurs after conjugation of two cells in most true yeast species, although some species may produce ascospores with or without conjugation.
• The yeast type is characterized by the presence of ascospores and the usual number of spores in each ascus.
• Ascospores can vary in color, texture, and shape.
• Fungi Imperfecti includes “false yeasts”, which do not produce ascospores and other sexual spores. Some yeast cells become chlamydospores through the formation of thick walls around them, such as Candida, Rhodotorula and Cryptococcus.

Cultural Characteristics

• The appearance of masses of yeast growth is not helpful in identifying yeasts. However, growth on the surface liquid media could indicate an oxidative yeast or film yeast. Carotenoid pigment production indicates the genus Rhodotorula.
• If the growth causes food to turn a shade of brown, it is important to see how it looks.
• It is not easy to distinguish yeast colonies from bacteria on agar plates. The only way to be certain is through microscopic examination.
• Young yeast colonies tend to be moist and slimy, although they can appear mealy. Most colonies are whitish but some colonies may be cream-colored or even pink.
• While some colonies are more stable with age than others, others become dry and wrinkled.
• Yeasts can be either oxidative or fermentative, but also both. The film yeasts are oxidative yeasts that form a film, pellicle or scum on the liquid’s surface. Fermentative yeasts are found in liquids and can produce cardon dioxide.

Physiological Characteristics

• Although yeast species may vary in their physiology, industrial yeasts have enough commonalities to allow generalizations. However, it should be remembered that exceptions will exist to every statement.
• Common yeasts thrive when there is plenty of moisture available.
• However, yeasts can grow in higher concentrations of solutes than bacteria. This means that they require less moisture than other bacteria.
• However, yeasts need more moisture than molds. Aw yeasts can be classified on the basis of water activity. They are considered ordinary if they don’t grow in high levels of solutes (i.e., low aw) and osmophilic, if they do.
• The lower limit of aw found in ordinary yeasts that have been tested so far is 0.88 to 0.94. Examples of minimal aw include 0.94 for beer yeast, 0.90 yeast made from condensed milk and 0.905 yeast for bakers.
• Osmophilic yeasts, however, have been observed growing slowly in media with an average aw of 0.62 to 0.65 for sirups. However, some osmophilic strains can be stopped at 0.78 in salt brine or sugar sirup.
• Each yeast is unique in its optimal aw value and the range of aw that it can grow under a particular environment. These aw values can vary depending on the nutritional properties of the substrate, pH and temperature, availability of oxygen, as well as the presence or absence inhibitory substances.
• Most yeasts can grow at temperatures similar to molds. The optimum temperature is between 25 and 30 C, while the maximum temperature is around 35 to 47 C.
• Some types can grow at 0 C and less.
• Most yeasts grow best in acidic environments, pH 4 to 4.5. They will not grow well in alkaline media unless they are adapted to it.
• The best conditions for yeast growth are aerobic, but fermentative yeasts can grow anaerobically, though they grow slowly.
• Sugars are generally the best energy source for yeasts. However, oxidative yeasts (e.g. the film yeasts) can oxidize organic acid and alcohol.
• The fermentation of yeasts produces carbon dioxide, which is used to leaven bread. Alcohol made from fermentative yeasts is used in the production of wine, beer and other beverages.
• The yeasts are also responsible for the production of flavor or “bouquet”, in wine.
• There are many types of nitrogenous foods, from simple compounds like ammonia or urea to complex compounds like amino acids and polypeptides.
• Additional growth factors are also required for yeast.
• The physiological characteristics of yeasts can change, particularly the true or ascosporeforming yeast, which has a sexual reproduction method.
• These yeasts may be bred to develop certain traits or may evolve into new forms.
• Many yeasts can adapt to conditions that would otherwise prevent them from growing well.
• Illustrative of different characteristics within a species is the large number of strains of Saccharomyces cerevisiae suited to different uses, e.g., bread strains, beer strains, wine strains, and high-alcohol-producing strains or varieties.

Classification and Identification of Yeasts

The true yeasts can be found in the Ascomycotina subdivision, while the false or asporogenous yeasts can be found in the Fungi Imperfecti and Deuteromycotina subdivisions. Barnett et. al. provides additional information about the identification and classification of yeasts.

Some yeasts can actually be found in two genera depending on their specific characteristics.

These are the main bases for the classification and identification of yeast genera:

1. How ascospores form.
2. If they are spore-forming
   1. Method of producing ascospores
      1. Produced without conjugation of yeast cells (parthenogenetically). Conjugation of ascospores and/or conjugation with small daughter cells may follow spore formation.
      2. Produced by isogamic conjugation (conjugating cell appearances similar).
      3. Heterogamic conjugation is used to produce heterogamic compounds (conjugating cells have different appearances).
   2. Ascospore appearance: size, color, shape. While most spores appear spheroidal, or ovoid, some spores have unusual shapes. For example, many species of Hansenula look like derby hats.
   3. There are usually one, two or four ascospores in each ascus.
3. Vegetative cells appearance: size, color and inclusions.
4. Method for asexual reproduction:
   1. Budding.
   2. Fission.
   3. Combination of fission and budding
   4. Arthrospores (oidia).
5. Producing a pseudomycelium or mycelium.
6. Growth of a film on the surface of a liquid (film yeasts), or throughout medium.
7. The color of macroscopical growth.
8. Physiological characteristics are used to distinguish species and strains within a species.
   1. Sources of carbon and nitrogen
   2. Vitamin requirements.
   3. Film yeasts can be fermentative or oxidative. Other yeasts could be fermentative, fermentative, and oxidative.
   4. Lipolysis, Urease Activity, Acid Production, or Formation of Starchlike Compounds.

Yeasts of Industrial Importance

The majority of yeasts used in industry are from the genus Saccharomyces. Any yeast that is not being encouraged or used in an industrial process is called “wild yeast”. Yeast used in one process could also be wild yeast in the next. Asporogenous yeasts, also known as false yeasts, are the most troublesome wild yeasts.

• Genus Schizosaccharomyces – These yeasts reproduce sexually by fission, form four to eight ascospores per ascus and are found in tropical fruits and soil. S. pombe is a common species.
• Genus Saccharomyces – These yeast cells can be round, oval, or elongated, and may form pseudomyceliums. Multipolar budding, ascospore formation or conjugation are methods of reproduction. Diploid cells may also be used to develop the embryonic stage. One to four ascospores are found per ascus. They are typically round or ovale. S. cerevisiae is the most widely used species. It produces top yeasts for beer, wine, and alcohol. Top yeasts are active fermenters, and can grow quickly at 20 C. The rapid evolution of CO2 and the clumping cells bring the cells to the surface. This is why the term “top yeast” was coined. Bottom yeast don’t clump and grow slower at lower temperatures (10-15 C). Because there is no clumping, and because CO2 has a slower growth rate and evolution, the yeast can settle to the bottom. This is why the yeast is called bottom yeast. These are just observations of yeast that brewers use. They do not explain why yeast can clump or flocculate. Rose and Harrison (1970) provide a detailed discussion of the theories underlying the flocculation in brewers yeasts. S. cerevisiae var. ellipsoideus is a high-alcohol-yielding variety used to produce industrial alcohol, wines, and distilled liquors. S. uvarum is a bottom yeast that is used to make beer. S. fragilis or S. lactis may be useful in making milk and milk products due to their ability to ferment lactose. S. rouxii, S. mellis and S. rouxii are osmophilic. Lodder (1984) states that many Saccharomyces were reclassified. S. uvarum, for example, is now considered to be a variant S. cerevisiae. S. fragilis, now Kluyveromyces marsxianus. S. lactis, now K. marxianus. S. rouxii and S. mellis are now Zygosaccharomyces rouxii. Debaryomyces kloeckeri now belongs to D. hansenii.
• Genus Kluyveromyces – These yeasts reproduce by multilateral budding and ascopores are released upon maturation. S. fragilis, S. lactis are discussed in the previous section.
• Genus Zygosaccharomyces: Some workers (Ludder 1984) consider this subgenus of Saccharomyces. These yeasts are known for their ability grow in high sugar concentrations (hence they’re called osmophilic). They are also involved in spoilage of honey, sirups and molasses as well as in the fermentation and soy sauce and certain wines. Z. nussbaumeri can be found in honey.
• Genus Pichia – These oval-to-cylindrical yeasts can form pseudomycelia. Ascospores can be hat-shaped or round and there are up to four per ascus. A pellicle forms when liquids are present. For example, P. membranaefaciens can form a pellicle by drinking beers or wine.
• Genus Hansenula – These yeasts look a lot like Pichia, but they are more fermentative than Pichia. However, some species can form pellicles. Ascospores can be hat- or Saturn-shaped.
• Genus Debaryomyces – These oval or round yeasts are made from pellicles in meat brines. Ascospores have an uneven surface. D. kloeckeri thrives on cheese and sausage
• Genus Hanseniaspora : This lemon-shaped (apiculate), yeast grows in fruit juices. Nadsonia yeasts grow in large, lemon-shaped containers.

False Yeasts (Fungi Imperfecti)

• Genus Torulopsis – These oval to round fermentative yeasts have multilateral budding and can cause problems in breweries. They also spoil many foods. T. sphaerica ferments milk products and lactose. Other species can spoil sweetened, condensed milk, juice concentrates and acid foods.
• Genus Candida: These yeasts are either pseudohyphae (or true hyphae) and have abundant blastospores or budding cells. They may also form chlamydospores. Many can form films and spoil foods high in salt and acid. C. utilis can be grown as food or feed. C. utilis has been grown with dairy starter cultures in order to increase the activity and longevity of the lactobacillus. Butter and oleomargarine can be spoilt by lipolytic C. lipolytica.
• Genus Brettanomyces – These ogive- and arch-shaped yeasts are responsible for the fermentation of late-fermented Belgian lambic beer as well as English beers. They are also found in French wines. B. bruxellansis is a typical species.
• Genus Kloeckera – These are imperfectly apiculate yeasts. K. apiculata can be found in fruits, flowers and soil.
• Genus Trichosporon – These yeasts form arthrospores and bud. They thrive at low temperatures, and can be found in breweries.
• Genus Rhodotorula – These yeasts can cause discolorations in foods such as colored spots on meats and pink areas in sauerkraut.

Groups of Yeasts

• Film yeasts (genera Pichia and Hansenula and Debaryomyces and Candida) grow on the surfaces of acid products like sauerkraut or pickles. They oxidize organic acids and allow less acid-tolerant organisms continue to spoilage.
• Hansenulas and Pichia can tolerate very high amounts of alcohol, and could oxidize it in alcoholic drinks.
• Pichia varieties are encouraged to grow on Jerez or Arbois wine. They are said to impart distinct flavors and esters.
• Debaryomyces can tolerate salty conditions and grow in cheese brines that contain as much as 24% salt.
• Film yeasts make little to no alcohol from sugars. Saccharomycodes and Hanseniaspora, Nadsonia and Kloeckera have lemon-shaped yeasts. They are considered to be objectionable for wine fermentations due to their high volatile acidity and low alcohol yields.
• Osmophilic yeasts (Saccharomyces rouxii, S. millis), thrive in environments of high osmotic tension, i.e. high concentrations sugars, salts and other solutes. This can cause spoilage of dried fruits, concentrated juices of fruit, honey, maple syrup, and other high-sugar options.
• Salt-tolerant yeasts can grow in salt brines, salted fish, curing brines, miso paste and tamari. The most salt-tolerant film yeasts are the species of Debaryomyces. They can be grown in curing brines, on meats and fish, and on cucumbers. Saccharomyces rouxii can also grow as a film on brine.
• Other genera of yeasts (Torulopsis and Brettanomyces) also grow in brines. Soy sauce has a high salt content (around 18 percent) and yeasts can grow in it.
• Saccharomyces rouxii, which is a yeast that produces alcohol and flavor, is a major player. However, other species such as Torulopsis and Pichia, Candida and Trichosporon may also grow. Sometimes, spoilage can occur when soy sauce is made from filmforming S. rouxii or Pichia. Miso production involves the use of similar yeasts, but different types will be used depending on the salt concentration.