Fungi and Food Spoilage || Yeasts

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<ul><li><p>Chapter 10</p><p>Yeasts</p><p>Yeasts are fungi which reproduce vegetatively by</p><p>means of single cells which bud, or less commonly,</p><p>divide by fission. This property enables yeasts to</p><p>increase rapidly in numbers in liquid environments,</p><p>which favour the dispersal of unicellular microor-</p><p>ganisms. Many yeasts grow readily under strictly</p><p>anaerobic conditions, again favouring their growth</p><p>in liquids. On the other hand, reproduction as single</p><p>cells restricts spreading on, or penetration into,</p><p>solid surfaces, where filamentous fungi are at an</p><p>advantage. Being eukaryotic organisms, yeasts</p><p>reproduce more slowly than do most bacteria, and</p><p>hence do not compete in environments which</p><p>favour bacteria, i.e. at pH values near neutral or at</p><p>very high temperatures. In common with filamen-</p><p>tous fungi, many yeasts are tolerant of acid condi-</p><p>tions. In broad terms, then, yeasts are more likely to</p><p>be active in acidic, liquid environments than else-</p><p>where. However, many yeasts also appear to be</p><p>highly resistant to sunlight and desiccation and</p><p>occur widely in nature on the surfaces of leaves,</p><p>fruits and vegetables.</p><p>When defined as above, i.e. as budding, nonpho-</p><p>tosynthetic eukaryotes, yeasts are a heterogeneous</p><p>assembly of often quite unrelated fungi. Barnett</p><p>et al. (2000) recognised almost 680 species of yeasts</p><p>in all, divided into over 90 genera: 57 were classified</p><p>as Ascomycetes, 36 as Basidiomycetes with 2</p><p>unclassified. It is not surprising, then, that yeasts</p><p>possess diverse properties.</p><p>As with other fungi, yeasts are classified into</p><p>genera primarily on the type and appearance of</p><p>spores, in this case ascospores or basidiospores.</p><p>Because yeasts possess limited morphological varia-</p><p>bility, classification at species level has traditionally</p><p>relied on biochemical tests, principally the utilisa-</p><p>tion of various carbon or nitrogen sources, and</p><p>vitamin requirements. Some physiological proper-</p><p>ties, i.e. growth at high temperature and reduced</p><p>water activity, are used in a secondary role.</p><p>The 2000 edition of Yeasts: characteristics and</p><p>identification listed 90 separate tests, including</p><p>utilisation of 47 carbon sources, 10 nitrogen</p><p>sources, vitamin requirements, fermentation pat-</p><p>terns, growth at various temperatures and in the</p><p>presence of elevated glucose and NaCl levels and</p><p>morphological characteristics. Clearly, taxonomy</p><p>of that kind is beyond the scope of the present work.</p><p>In recent years, DNA sequencing has become a</p><p>major classification tool and is now commonly used</p><p>for yeast identification. Molecular methods have</p><p>clarified some of the bases for taxonomy of yeasts,</p><p>but at the same time have led to increasing complex-</p><p>ity, with a seemingly random reassortment of spe-</p><p>cies into genera with each new monograph.</p><p>Teleomorphanamorph connections. As with</p><p>other fungi of industrial significance, ascomycetous</p><p>and basidiomycetous yeasts frequently reproduce in</p><p>nature or in the laboratory as their anamorphic</p><p>states, and hence possess anamorphic names. Dili-</p><p>gent study has in recent years enabled yeast specia-</p><p>lists to linkmany anamorphs with teleomorphs, and</p><p>in many cases the anamorph name has fallen into</p><p>disuse. As with some filamentous fungi, however, a</p><p>case for dual nomenclature exists because establish-</p><p>ing the anamorphteleomorph connection is often</p><p>difficult. Sometimes too the link is tenuous, with</p><p>only a few isolates carrying the life cycle to comple-</p><p>tion. Dual names have been retained here for most</p><p>species discussed.</p><p>J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_10, Springer ScienceBusiness Media, LLC 2009</p><p>357</p></li><li><p>Yeasts in foods. By comparison with many</p><p>strictly filamentous fungi, yeasts possess limited</p><p>biochemical pathways and quite fastidious nutri-</p><p>tional requirements. Foodstuffs, generally substrates</p><p>rich in the hexose sugars, minerals and vitamins,</p><p>which many yeasts require for growth, are an ideal</p><p>substrate for yeasts. Their association with the phyl-</p><p>losphere of many crops ensures their presence on</p><p>fruit and vegetables, and their entry into food proces-</p><p>sing plants.</p><p>Relatively few yeast species cause significant</p><p>spoilage in processed foods, most being adventi-</p><p>tious contaminants from natural sources. Deak</p><p>and Beuchat (1996) list 99 yeast species which</p><p>occur in foods including fruit, beverages, wine,</p><p>beer, meat, dairy products, low aw products and</p><p>low pH products. Many of these yeasts grow poorly</p><p>if at all in properly formulated processed foods,</p><p>as they are intolerant of reduced water activity,</p><p>heat processing or preservatives. Even if limited</p><p>metabolism by such yeasts does occur, it is usually</p><p>of little consequence unless there is significant gas</p><p>production. It is doubtful whether any yeasts pro-</p><p>duce mycotoxins, and few produce even marginally</p><p>unacceptable off-odours. There are, of course,</p><p>exceptions. Certain species must be classified as</p><p>spoilage yeasts because they possess one or more</p><p>undesirable properties. These commonly occurring</p><p>spoilage species form the basis for most of the sub-</p><p>ject matter of this chapter.</p><p>Spoilage yeasts. In our experience, only about ten</p><p>species of yeasts are responsible for spoilage of</p><p>foods which have been processed and packaged</p><p>according to normal standards of good manufac-</p><p>turing practice. These species are responsible for</p><p>major losses of processed foods around the world</p><p>every year. We believe that relatively simple tests</p><p>can be used to identify them when taken in conjunc-</p><p>tion with their spoilage habitats. These species are</p><p>listed in Table 10.1, with their principal undesirable</p><p>properties. Two species which do not cause spoi-</p><p>lage, but are of widespread occurrence in foods, are</p><p>also included in Table 10.1, and in subsequent</p><p>descriptions in this chapter.</p><p>It is important to note that if good manufactur-</p><p>ing practice is neglected, i.e. if factory hygiene is</p><p>poor, if preservatives are omitted, either deliber-</p><p>ately or unintentionally, if pasteurising tempera-</p><p>tures are inadequate, or filling machinery or factory</p><p>premises are unsanitary, raw materials are of poor</p><p>quality, brining or syruping procedures are poorly</p><p>controlled, etc., many other adventitious yeast con-</p><p>taminants can develop in a product. In such cases</p><p>the following text will be of little value and neither</p><p>will identification of the yeasts concerned. The cor-</p><p>rect approach, and often the only recourse in such</p><p>Table 10.1 Spoilage yeasts</p><p>Yeast Important properties</p><p>Brettanomyces bruxellensis Production of off-odours in beer, cider and soft drinks</p><p>Candida krusei Preservative resistant; film formation on olives, pickles and sauces</p><p>C. parapsilosis Lipolysis, fermentative; causes spoilage in a wide range of foods including cheese, margarines,dairy and fruit products</p><p>Debaryomyces hansenii Growth at low water activities in foods preserved with NaCl, especially salt meat</p><p>Kazachstania exigua Moderately preservative resistant; of common occurrence in olive brines, relevantly rarely thecause of spoilage of sauerkraut or of juices, dairy products or soft drinks</p><p>Kloeckera apiculata Spoilage of fresh and processed fruit</p><p>Pichia anomala Spoilage of processed fruit and yoghurts containing fruit</p><p>P. membranaefaciens Preservative resistant; film formation on olives, pickles and sauces</p><p>Rhodotorula mucilaginosaR. glutinis</p><p>Common food contaminants; spoilage of dairy products; occasional spoilage of fresh fruits</p><p>Saccharomyces cerevisiae Common food contaminant; sometimes fermentative spoilage of soft drinks; some strainspreservative resistant</p><p>Schizosaccharomyces pombe Preservative resistant; relatively rare spoilage yeast</p><p>Zygosaccharomyces bailii Preservative resistant; fermentative spoilage of acid, liquid preserved products such as juices,sauces, ciders and wines</p><p>Z. bisporus Preservative resistant; properties intermediate between Z. bailii and Z. rouxii</p><p>Z. rouxii Growth at extremely low water activities; fermentative spoilage of juice concentrates, honey,jams, confectionery, packaged dried fruits, etc.</p><p>358 10 Yeasts</p></li><li><p>cases, is to pay attention to manufacturing guide-</p><p>lines, so that this kind of problem is positively</p><p>eliminated.</p><p>Identification of spoilage yeasts. Considerable</p><p>work has been carried out in recent years to develop</p><p>simplified systems for identification of foodborne</p><p>yeasts, as yeast identification procedures based on</p><p>biochemical reactions are too complex and take too</p><p>long to be of value in the food or industrial micro-</p><p>biology laboratory. Several automated systems are</p><p>now commonly used, as well as simpler laboratory-</p><p>based systems, and these are discussed in some</p><p>detail in Chapter 4. Identification using DNA</p><p>sequencing is increasingly becoming the method of</p><p>choice, as extensive databases such as GenBank are</p><p>freely available for identification purposes. The</p><p>600650 nucleotide D1/D2 region of the large sub-</p><p>unit (26S) ribosomal DNA is the most widely tar-</p><p>geted section of the genome, and sometimes the ITS</p><p>region may also be used (Kurtzman et al., 2003).</p><p>However, a word of caution. Results presented by a</p><p>computer identification system or DNA sequence</p><p>database still need to be interpreted with care: com-</p><p>puters are not infallible, and sequences in DNA</p><p>databases do not always have the correct species</p><p>name! Does the result make sense? Keep in mind</p><p>the source of the yeast isolate and the spoilage pro-</p><p>blem being investigated. Is the yeast reported a</p><p>relatively common foodborne yeast, or a rare yeast</p><p>only ever isolated once or twice from some obscure</p><p>source? If the computer key gives a list of yeasts</p><p>differing by one result (one discrepant test), check</p><p>this list along with the discrepant test to see if your</p><p>yeast is really more likely to be one of those listed as</p><p>a second option. A weak reaction may have been</p><p>incorrectly interpreted as negative. Perhaps carry-</p><p>ing out one or two other tests can give better results.</p><p>A simplified approach has been used here which</p><p>differentiates the species of yeasts most commonly</p><p>responsible for actual spoilage of processed foods</p><p>and beverages. Table 10.2 lists a series of media and</p><p>conditions which will enable differentiation of the</p><p>species listed in Table 10.1. To simplify and expedite</p><p>identification as far as possible most media and con-</p><p>ditions specified here have been used in the identifi-</p><p>cation of filamentous fungi elsewhere in this book.</p><p>The exceptions are the use of malt acetic agar, Cza-</p><p>pek agar and growth on MEA at 378C. The first ofthese media distinguishes preservative-resistant</p><p>yeasts from others; on the second only yeasts which</p><p>utilise nitrate as a sole carbon source can grow.MEA</p><p>is used at 378C rather than the customary CYAbecause some yeasts grow poorly on CYA. Czapek</p><p>agar is made with the same ingredients and proce-</p><p>dures as CYA but yeast extract is omitted.</p><p>It is emphasised that the procedures used here will</p><p>work only for yeasts causing actual spoilage: the</p><p>system is not designed to cope with the myriad spe-</p><p>cies of yeasts than can occur adventitiously in foods.</p><p>Even so, the procedures are not rigorous and may on</p><p>occasion misidentify an excluded yeast as being one</p><p>of those discussed here. Provided all tests are carried</p><p>out as specified and morphological observations</p><p>made, such occasionswill be infrequent. In particular</p><p>preservative-resistant yeasts, the most important</p><p>yeasts in food spoilage will usually be readily recog-</p><p>nised by the techniques described below.</p><p>Procedures for yeast identification. Pour Petri</p><p>dishes with the media listed in Table 10.2. From a</p><p>3 to 7 day old slant culture of the yeast, preferably</p><p>growing onMEA, disperse a small loopful of cells in</p><p>35 ml sterile water or 0.1% peptone. Streak each</p><p>plate with a loopful of cells from this inoculum. A</p><p>suitable streaking technique is described in Chapter</p><p>4. Incubate plates for 3 days, then examine each for</p><p>presence or absence of growth. Note also colony</p><p>colour and the size and shape (regular or irregular)</p><p>of well separated colonies. Make a wet microscopic</p><p>mount in water, lactic acid or lactofuchsin (see</p><p>Table 10.2 Media and conditions for identification ofspoilage yeastsa</p><p>Medium Purpose</p><p>Czapek agar Assessing ability to utilise nitrateas a sole nitrogen source</p><p>Malt Extract Agar(MEA)</p><p>Colony and cell morphology</p><p>Malt Acetic Agar(MAA)</p><p>Assessing preservative resistance</p><p>MEA at 378C Assessing growth at elevatedtemperatures</p><p>Malt Yeast 50%Glucose Agar(MY50G)</p><p>Assessing growth at reducedwater activities in the presenceof high carbohydrate levels</p><p>Malt Yeast 10% Salt12% Glucose Agar(MY10-12)</p><p>Assessing growth at reducedwater activities in the presenceof high sodium chloride</p><p>a Incubation is at 258C unless specified. Inspection should beat 3 and 7 days after inoculation.</p><p>10 Yeasts 359</p></li><li><p>Chapter 4) from the MEA plate grown at 258C andfrom malt acetic agar. Record approximate cell size</p><p>and shape, position of budding and the presence or</p><p>absence, type and number of ascospores (see the</p><p>following figures for a guide). Reincubate plates</p><p>and repeat observations at 7 days.</p><p>Salient properties on these media of yeasts</p><p>included here are listed in Table 10.3. The key which</p><p>follows is based on growth for 7 days on the media in</p><p>Table 10.2. Cell sizes are from colonies on MEA at</p><p>258C, aged between 3 and 7 days. The species aredescribed and discussed below in alphabetical order.</p><p>Brettanomyces bruxellensis Kuff. &amp; van LaerFig. 10.1</p><p>Brettanomyces intermedius (Krumbholz &amp; Tauschan.) van der</p><p>Walt &amp; Keuken</p><p>Dekkera intermedia van der Walt</p><p>Teleomorph: Dekkera bruxellensis van der Walt</p><p>Colonies on MEA at 3 days minute, white; at 7 days</p><p>1.52.0 mm diam, white, convex, margins circular,</p><p>surface glistening. Cells on MEA at 3 days mostly</p><p>ellipsoidal to ogival (pointed at one end, rounded at</p><p>the other), less commonly spherical or cylindrical,</p><p>4.57 3.04.0 mm, reproducing by budding, term-inally or subterminally, but not laterally; occurring</p><p>singly, in pairs, short chains or clusters.</p><p>Teleomorph not produced under conditions used</p><p>here. An acetic acid or other sharp or fruity off-</p><p>odour usually produced.</p><p>No growth onCzapek agar; growth at 378Cmorerapid than at 258C; no growth on malt acetic agar;slow growth on MY50G; no growth on MY10-12.</p><p>Distinctive features. Species of Brettanomyces are</p><p>distinguished by the formation of ogival cells, exclu-</p><p>sively terminal budding and the production of acetic</p><p>acid from glucose under aerobic conditions. Cul-</p><p>tures are usually short lived unless 2% calcium car-</p><p>bonate is incorporated in the growth medium to</p><p>neutralise the acid produced.</p><p>MostBrettanomyces species have similar properties;</p><p>identification of an isolate to genus is usually sufficient.</p><p>B. bruxellensis, in our experience and that of others, is</p><p>the species most commonly isolated from foods.</p><p>Taxonomy.Kurzman and Fell (1998) and Barnett</p><p>et al. (2000) agree thatBrettanomyces intermedius is a</p><p>synonym of B. bruxellensis.</p><p>Physiology. As noted above, the most important</p><p>characteristic ofBrettanomyces bruxellensis and other</p><p>Brettanomyces species is the ability to produce acetic</p><p>acid from glucose. Pitt (1974a) reported growth of</p><p>this species down to pH 1.8 in a medium acidified</p><p>with HCl and to pH 2.3 in citri...</p></li></ul>

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