Spoilage yeasts with emphasis on the genus Zygosaccharomyces

6

Spoilage yeasts with emphasis on the genus Zygosaccharomyces STEPHENA. JAMESand MALCOLM STRATFORD

6.1

Introduction

Yeasts, as a group of microorganisms,are well known for their positive contributions in the production of a variety of different foods and beverages, including beer, bread, chocolate, cider, sakt, soy sauce and wine. Indeed, Saccharomyces cerevisiue is perhaps the best-recognised yeast species for such purposes, and is often referred to simply as either baker’s or brewer’s yeast, depending in which process it is being used (i. e., baking or brewing). However, as well as having a positive role in food and beverage production, yeasts can also have a negative role, with a limited number of species (including S. cerevisiue) noted for their ability to cause spoilage.

In nature, yeasts are found in a variety of different habitats, including soil, freshwater, salt water and air [43]. Likewise, these predominantly unicellular fungi can also be found associated with many different animals, insects (e. g., Drosophilu spp.) and plants (especially fruit and vegetables) [6]. Consequently, due to their widespread distribution, and presence on the surfaces of many fruit and vegetables [13], yeasts are routinely encountered as contaminants in a variety of different foods and beverages, including alcoholic and soft drink beverages, bakery products, dairy products (e. g., yoghurt and cheese), fresh and processed fruits and vegetables, fresh and processed meats and seafood 16, 19,44,56,78]. Indeed in a recent survey by BARNEITet al. [6], of the 678 listed yeast species, more than 150 species from over 40 different genera (representing approx. half of all currently accepted genera) were noted as having a foodheverage association. Typically yeasts become the dominant contaminantsof foods and beverages when competition from other microorganisms,particularly bacteria and moulds, is restricted by low pH, presence of preservatives (e. g., benzoic and sorbic acids), high sugar and/or alcohol concentrations [56]. Yeasts have the ability to grow or cause spoilage in foods at near neutral pH, but rarely do so, following the thermal treatments given to “low acid foods” to destroy bacterial pathogens. However, despite the number of yeast species recorded as being foodmeverage contaminants, very few are in fact viewed as significant spoilage species. Indeed, in their recent review of fungi associated with food spoilage, P ~ and T HOCKING[56] listed a total of only 12 yeast species (Table 6.1-1) which they saw as being responsible for causing the vast majority of spoilage incidents by yeasts of foods and beverages prepared according to normal standards of good manufacturing practice (GMP). In a ’forensic approach’ to spoilage of soft drinks, DAVENPORT [lo, 111 noted that most yeast contaminants encountered could be categorised as either opportunistic spoilage species (Group 2) or indicators of poor factory hygiene (Group 3). Unlike many of the significant (Group 1) spoilage yeasts listed in Tab171

Detrimental aspects of Zygosaccharomyces Table 6.1-1 Yeast species most commonly associated with food and beverage spoilage (adapted from [56]). Species

Spoilage properties

Brettanomyces bruxellensis (= Dekkera bruxdlmsis)

productionof off-odours'

Candid holmii (= Sacchammyces exiguus)

moderately preservativeR

Candida krusei (= lssatchenkiaorientalis)

preservativeR,film forming

Can@& parapsilosis

lipolytic, fermentative

Debwyomyces hansenii Kloeckera apiculata (= Hanseniaspora uvarum) Pichia menbranifaciens Sacchammyces cerevisiae Schizosaccharomyces pombe

osmotolerant fermentative preservativeR,film forming fermentative, preservativeR" preseNative

Zygosaccharomyces bailii

fermentative, osmotolerant, preservativeR

Zygosaccharomyces bisporus

fermentative, osmotolerant, preservativeR

Zygosaccharomyces muxii

fermentative, osmotolerant preseNative

Teleomorphic (i. e., sexual) species names are highlighted in bold type. Due to productionof acetic acid ** Some strains only resistance

le 6.1-1, opportunistic spoilage species generally only give rise to spoilage if processing errors occur, such as under-dosing or omission of preservatives, gross errors in hygiene or use of inadequate pasteurisation temperatures. Key characteristics noted by DAVENPORT[lo, 11, 121 that distinguished the major spoilage species, such as 2. builii, from opportunistic spoilage species included preservative resistance, osmotolerance, and high fermentative ability. Group 2 opportunistic spoilage yeasts of soft drinks included Candidapurupsilosis, Fichiu membranijiaciens and Deburyomyces hansenii (= Candidafamutu) [71]. In contrast, poor-hygiene-indicatorspecies (e. g., Aureobasidium pulluluns and Rhodotomla spp., indicators of dust contamination when found in soft drinks) are generally incapable of causing spoilage, even when processing errors do arise and allow significant infection.

6.2

Detrimental aspects of

Zygosaccharomyces

To the food and drinks industries, the most problematic spoilage yeasts encountered are undoubtedly those belonging to the genus Zygosucchuromyces.Indeed this genus is often re172

Table 6.2-1 Species belongingto the genus Zygosaccharomycesand typical food productsfrom which they have been recovered [adaptedfrom refs. 6,33, 42,64, and 751.

species Z. bailii

Z. bisporus Z cidn Z. fermenfati Z. florentinus Z. kombuchaensis Z. lentus Z. mellis Z. microellipsoides Z. mrakii Z rouxii

Foods isolated from Apple juice, confectionery, fitness drinks, grape and blackcurrant juice, grape juice, honey, marzipan, mayonnaise, orange-juice concentrate, pickles, salad cream, sorghum-brandy mash, wine, Worcester sauce Fennentingcucumbers, orange drink Cider Cola drink, orange drink, strawberry drink Cola drink, lemonade, orangeade, soda water Kombucha tea Orange squash, tomato ketchup, whole-orange juice, wine Honey, strawberry juice, sugar, Apple juice, lemonade None recorded

Candied fruit, cane sugar, chocolate filling, fruit juice concentrates

(e. g., apple, orange), honey, jam, maple syrup, marmalade, marzipan, molasses, orange syrup, soft drinks, soy sauce, sugar syrup, wine

garded as being synonymous with food spoilage. Currently the genus comprises of 11 species (Table 6.2-l), three of which, namely Z. bailii, Z. rouii and to a lesser extent Z bisporus are well recognised as representing significant food and beverage spoilage organisms (Table 6.1-1). Key physiological characteristics that make these yeasts such problematic spoilage agents include significant resistance to weak acid preservatives (notably Z bailir?, extreme osmotolerance (notably Z rouii and Z mellis), and ability to vigorously ferment hexose sugars such as glucose and fructose (i. e., most members of the genus). Recently, STEELS et al. [@] using ribosomal DNA (rDNA) sequences identified a new Zygosaccharomyces species, Z lentus, which was found to have similar spoilage characteristics to those possessed by Z. bailii, Z bisporus and Z. rouxii (i. e., osmotolerance and preservative resistance) [65].Furthermore, unlike Z. builii, Z lenrus was also shown to be able to grow (albeit slowly) at low temperature (i. e., 4 "C), suggesting that this species might pose a threat as a potential spoilage organism of chilled food products. Foods at particular risk to spoilage by these yeasts tend to be acidic (pH 2.5 to 5.0) and contain high concentrationsof fermentable sugars f751.Foods commonly spoiled include h i t juices, juice concentrates, soft drinks, sugar syrups, jams and preserves, honey, tomato sauce, mayonnaise and wines [6,38,75]. With regard to the types of spoilage that yeasts cause, this is very much dependent upon the food or beverage that they are found in. Typical spoilage includes generation of taints, 173

Detrimentalaspects of Zygosaccharoqces

odours and off-flavours, development of hazes, and excessive gas production. Clouds and hazes generally refer to the appearance of substantive yeast growth, which is barely visible at 1 6 cells/ml, but may reach lo7 celldml. Such growth may not be visible in cloudy foodproducts, such as fruit juices, but 40 % of commercial fruit juices in Tetrapaks were found to contain viable yeasts including Z rorurii and Z bailii [141. On occasion yeast growth has the appearance of particulates, usually as a result of pseudohyphal formation or flocculation. Spoilage of ciders by Saccharomycodes ludwigii often results in 1-3 mm sized particles. Alternatively, surface-films can be produced, notably by Pichia mernbranifaciens or Zssatchenkia orientalis (= Candida krusez] [56,801, and on occasion by Zygosaccharomyces [35,72]. In more solid foods, yeast spoilage usually takes the form of unsightly colonial surface growth, although gas bubbles can form within the food by fermentation. Where substantial yeast growth has occurred, it is likely that other attributes of yeast spoilage will also become evident. It is rare for spoilage flavours, odours or taints to occur without yeast growth to at least 16cells/ml. ~ r ~ woft fermentative h yeast may result in gas production and also ethanol, imparting a sweet alcoholic taste. Off flavours and taints may also be caused by microbial secondary metabolite formation, following growth of substantial yeast populations (e. g., ethyl acetate [44]).Alternatively yeast enzymes acting on components of the food may also result in unpleasant taints. As an example, certain spoilage yeasts and moulds are able to decarboxylate sub-inhibitory levels of the preservative sorbic acid (2,4-hexadienoic acid) to form 3,5-pentadienewith a characteristic kerosene smell. Excessive gas production is a direct and noticeable consequence of high fermentative ability in spoilage yeasts. It may be speculated that a significantly higher proportion of foods spoiled by fermentative yeasts result in consumer complaints, as compared with spoilageby non-fermentativeyeasts. Indeed, if left unchecked, yeast fermentation in a food or beverage (i. e., fermentative spoilage) can generate very significant amounts of CO2, which in turn can lead to the distortion or explosion of either the product (e. g., fondant-filled chocolate cream eggs) or the product packaging. While none of the spoilage-associatedZygosaccharomyces species are known to be pathogenic to man, TODD[77]nevertheless reported several cases where yeast ingestion was believed to have caused gastrointestinal disorders. Similarly, product spoilage by Zygosaccharomyces species can lead to serious injury (e. g., eye damage), particularly if glass bottles are caused to explode [21].

6.3

Physiological background of spoilage by Zygosaccharomyces

The key physiologicalcharacteristicthat make the Zygosaccharomycessuch notorious spoilage yeasts are their resistance to the preservatives commonly used in the food industry. Preservativeresistancehas been widely studied in yeast but in recent years this has predominautly involved the moderately preservative-resistantyeast S. cerevisiae, due to its role in the pioneering of genetic manipulation and subsequent genome sequencing [20]. It is commonly 1 74

Detrimentalaspects of 2)gooaccharomyces

assumed that many of the mechanisms of resistance identifiedin S. cerevisiue will have similar counterparts in the more resistant Zygosucchuromyces yeasts. The preservatives examined are those most commonly used in foods: sorbic and benzoic acids used in beverages, processed fruit, fish, vegetables, spreads, sauces and confectionery; acetic acid in pickles, sauces, dressings and vinegars; propionic acid in bread; sulphite (Sod in wines and cider. These are often collectivelytermed the “weak-acid preservatives”.Zygosucchuromycesspp. yeasts are known to be phenomenally resistant to sorbic, benzoic, propionic and acetic acids, and have the capacityto adapt to higher concentrations[26,49,82,83,84]. KABARA and EKL~IND[34] have reviewed the antimicrobialnature of these acids and their use in foods. Weak acid preservatives are fungistatic rather than fungicidal and are commonly thought to inhibit growth of yeasts by acidification of the cytoplasm. Weak acids in aqueous solution form dynamic equilibria between undissociated acid molecules and their respective charged anions, with acid molecules predominating at low pH. It is known that uncharged weak acid molecules dissolve easily in lipids and are able to pass rapidly through the plasma membrane by Fick-type diffusion [9,27,62,69,85]. The classical “weak acid theory” proposes that uncharged acid molecules, on passing into the neutral pH of the cytoplasm, are forced to dissociate, thus accumulating as charged anions within the cytoplasm, and simultaneously releasing protons. Excessive proton release overcomes cytoplasmicbuffering and causes acidificationof the cell, inhibiting key enzymes and preventing active transport. This theory was independently proposed to account for the action of 2,4dinitrophenol[36], acetic acid [48], sulphite [67], sorbic acid (A. WARTH,pers. commun.) and benzoic acid [37]. Cytoplasmic acidification has been experimentally verified in S. cerevisiue for acetic acid [48], benzoic acid [37l, and in both S. cerevisiue andZ builii for sulphite [53]. Other mechanisms of action for preservatives have been proposed and are reviewed by KABARA and E K L W [34]. More recent work has suggested a membrane disrupting action for sorbic acid 1681 and involvement of benzoic acid in glycolysis [Sl]. Microbial mechanisms of resistance can, in broad terns, be summarized as three possible strategies: i>destruction of the inhibiting agent; ii) prevention of entry, or removal, of the inhibitor from the cell; iii) alteration of the inhibitor target, or amelioration of the damage caused. All three strategieshave been suggested in accounting for yeast resistance to weakacid preservatives. Sulphite removal by production of binding agents was shown to be a key factor prior to growth of S. cerevisiue and Succharomycodes Zudwigii [67, 701. The ability of Z builii strains to metabolize acetate in the presence of glucose may contribute to the resistance of this yeast to acetic acid [63], by removing acetic acid and thereby raising the pH of the cell cytoplasm [75]. Metabolism of benzoic acid by Z builii via p-hydroxybenzoicacid and catecho4 was reported by INGRAM [26], and recently the molecular mechanism for this transformation was elucidated, with the ZbyME2 gene shown to confer the ability to metabolize both sorbate and benzoate [46]. However, other studies have concluded that metabolism of weak acids is of limited benefit and insufficient to account for the resistance of Z builii to preservatives [26, 66, 821. 175

Detrimental aspects of Zypsacchmmyces

-

Prevention of entry, or removal of acidic preservatives was suggested by observations that resistant yeasts showed slower permeation by benzoic or propionic acids [85].WARTHproposed that in Z. bailii, an active “sorbate pump” conferred resistance by extrusion of preservatives [82,83,84,85]. This view was disputed by observations that the distribution of weak acids was exactly as predicted from the pH of media and cytoplasm [7, 81. However, in S.cerwisiae active efflux of benzoate was suggested as a resistance mechanism [22], a view strengthened by the discovery that deletion of any of a number of major facilitator superfamily transporters, increased sensitivity to preservatives. These included PDRI2 for sorbate, benzoate andacetate [25,55], SSUI for sulphite [50], andAZRI for acetic acid [73]. Amelioration of the damage caused by preservatives (i. e., low pH cytoplasm), involves removal of acid from the cell. The plasma membrane p-ATPase ejects protons from the cell in an energy-dependent manner, and has been shown to aid resistance to acetic and sorbic acids [24,79]. Hsp3O may also affect weak-acid resistance via regulation of the H+-ATPase

P41. In light of these reports, these characteristics will be discussed in more detail in context of each of the three main Zygosaccharomyces spoilage species (i. e., Z. bailii, Z. bisporur and Z.rouxii), as well as the recently described Z. lenrus [64].

6.3.1

Zygosaccharumyces bailii

Among the genus Zygosaccharomyces, Z bailii undoubtedly represents the most problematic spoilage yeast to the food and beverage industries. The reason for this is that one of the most distinctive physiological characteristic of this species, from a spoilage perspective, is its exceptional resistance to weak acid preservatives such as acetic, benzoic, propionic and sorbic acids, which are commonly used in the preservation of foods and beverages. Individual cells in populations of Z. bailii have been shown to vary considerably in their resistance to sorbic acid, with rare “super cells” able to grow in levels of preservative double that of the average population [66]. Often, Z bailii strains are recovered (from spoiled food products) whose resistance to these types of preservatives far exceeds the levels legally permitted in Europe (i. e., 300 mg I-’ in soft drinks [2]). Besides displaying phenomenal resistance to these types of preservative, Z. bailii is also remarkable for the fact that exposure to low, sub-inhibitory levels of this type of preservative can often lead to adaptation. As a result, strains of this species can gain the ability to both survive and proliferate in concentrations of (weak acid) preservative much higher than prior to adaptation. Besides being preservative resistant, other features that contribute to the spoilage capacity of Z bailii are: i) its ability to vigorously ferment glucose, ii) ability to cause product spoilage from an initial inoculum of as little as one viable cell per litre or package {lo, 171 and iii) moderate osmotolerance (in comparison to Z. rouxii). Like S.cerevisiae, Z. bailii can continue to ferment glucose under significant atmospheric pressure [28], but unlike S.cerevisiae, Z. bailii can do so in the presence of high levels of preservative. Indeed, sugar fer176

Detrimental aspects of Zygcwaccharomycas

mentation by Z bailii and the Zygosaccharomyces species in general is quite unique, as unlike other yeasts, these species appear to metabolize fructose in preference to glucose (a phenomenon referred to as fructophily) [ 151. Consequently,the growth rate of Z bailii in a food product is often enhanced if the level of fructose present exceeds 1 % of the total product composition. Although not as osmotolerant as Z rouxii (see following section), Z bailii is nevertheless still capable of growing at a water activity (&I) as low as 0.80 at 25 "C [56].

6.3.2

Zygosaccharomyces bisporus

Genealogically,Z. bispoms has been shown by ribosomal DNA (rDNA) sequence analysis to be closely related to Z builii [29]. Consequently, this species shares many of the physiological characteristics possessed by Z bailii [6], including preservative resistance and 0smotolerance, which can sometimes hamper accurate identification based on conventional physiological methods between isolates of these two species [88]. However, in contrast to Z builii, Z. bispoms is reported to be marginally more osmotolerant [76]. While individual strains show considerable variation, Z bispoms is on the whole slightly less resistant to weak acid preservativesthan Z. bailii (H. STEELS, pers. commun.). From a spoilageperspective, despite having similar physiological characteristics to Z bailii, Z bispoms probably does not represent the same sort of (spoilage) threat as Z. bailii, as it does not appear to be as widely distributed as Z. bailii [6].

6.3.3

Zygosaccharomyces lentus

Recently, a spoilage yeast was isolated from spoiled whole-orange drink, and initially identified by conventional methods [6, 891, as Z bailii. However, closer physiological examination of this isolate revealed it to possess atypical Z builii characteristics, displaying extremely poor growth when grown aerobically in shake culture, and failing to grow in the presence of 1 % acetic acid (a diagnostic test used to distinguish Z. bailii and 2. bisporus from other Zygosaccharomyces species [6,38]). Subsequent 18s rDNA sequence analysis of this isolate, as well as four additional physiologically atypical Z. bailii strains, revealed the existence of a phylogenetically distinct taxon, closely related to, but nevertheless separate from, 2 bailii and Z bisporus [MI. Based on its slow growth under aerobic conditions, STEELS et al. [64] named this novel Zygosaccharomyces species Z. lentus. Figure 6.3-1 shows a scanning electron micrograph of Z. lentus IGC 5316, a strain isolated from spoiled wine [641, As a consequence of the close genealogical relationship exhibited between these three Zygosaccharomyces spp. (Fig. 6.3-2), Z lentus was found to share many of the spoilage characteristics possessed by both 2: bailii and Z bisporus, including osmotolerance (i. e., ability to grow in the presence of 60 % [w/v] sugar), resistance to weak acid preservatives Vel(e. g., benzoic and sorbic acids), as well as resistance to dimethyldicarbonate(DMDC, 177

Detrimental aspects of zLgosaccharomyces

Fig. 6.3-1 Scanning electromicrograph oi Z lentus IGC 5316, isolated from spoiled wine [64] (do Mark Kirkland, Unilever Research Colworth Laboratory)

corin Tm) a sterilant occasionally used in the beverage indusmes [64,65]. To date, nine isolates of Z. lentus have been identified and characterized, of which five are known to have originated from spoiled food products (i. e., orange squash, orange juice, tomato ketchup and wine [ a ] ) . However, unlike both Z. builii and Z. bisporus, Z. lentus will grow (albeit slowfy) at low temperature (i. e., 4 "C).Such ability therefore raises the possibility that this recently described Zygosuccharomyces species could pose a real threat to the food and drinks industries as a spoilage agent of chilled products [ a ] .

6.3.4

Zygosaccharomyces rouxii

As a spoilage organism, Z. rouii is physiologically distinctive as being the most osmotolerant yeast species known. Indeed, of all known organisms only the filamentous fungus Xeromyces bispoms displays greater osmotolerance than Z. rouxii [56]. This yeast has been recorded as being able to grow at a water activity (a,) of 0.62 in fructose and 0.65 in sucrose/glycerol[76].Consequently,Z. rouii is particularly noted for its spoilage of raw cane 178

Detrimentalas-

Z. lentus NCYC 2789T

87 84

-

of Zcraosacchammwcas

Z. b i p r u s NCYC 1495T

Z. bailk‘NCYC 1416T

0.01

Fig. 6.3-2 Dendrogram showing the phylogenetic relationship between members of the ~ p s a c c h a r o ~ c esensu s strict0 species group I421 based on 26S rDNA D1m2 gene sequences. Those ZLgOsaccbmmycas species noted for their abilily to cause spoilage are highlighted in boldfont. The tree was constructed by usingthe neighbour-joiningmethod [SS].Bootstrap values, expressed as percentages of 100 replications, are given at branch nodes (only values > 50 % are shown). Scale bar, 1-estimatedbase substitution per 100 nucleotides.

sugar and food products of high sugar content, such as fruit juice concentrates,honey, jam, marzipan and sugar syrups [6,33,76]. Besides being osmotolerant,Z. rouxii is also preservative resistant; though not to the same degree as Z badii, and like all Zygosuccharomyces will vigorously ferment hexose sugars (e. g., glucose). Thus,as a causative agent of fermentative food spoilage, Z rouxii ranks second only to Z builii [56].

6.3.5

Other Zygosaccharomyces spoilage species

Other, less well-known members of the genus Zygosucchuromyces can also cause spoilage of foods. These include 2 cidri, Z fermentuti, Z florentinus, Z mellis, and Z. microellipsoides (Table 6.2-1). Z mellis, frequently confused with and mistaken for Z. rouxii, is perhaps best known for causing spoilage in honey. Z microellipsoides has been described as “less dangerous than S. cerevisiue” [59] and is closely related to Toruluspora delbrueckii [30]. Both T. delbrueckii and Z. microellipsoides are fermentative [6], osmotolerant [60], and widely distributed in soft drinks plan^. Both species are capable of causing spoilage, but are relatively sensitive to preservatives. Z cidri, Z. fennentati and Z. florentinus are all

179

Detrimental aspects of Zypsaccharomyces

moderately osmotolerant, fermentative species, capable of causing spoilage. They are generally more sensitive to preservatives than s. cerevisiue (H. STEELS, pers. C O ~ U and ~ . ) less prone to cause spoilage.

6.4

Specific methods to study spoilage by Zygosaccharomyces

In order to prevent, or at the very least reduce the likelihood of product spoilage by yeasts, and particularly by osmotolerant and preservative resistant species such as Z builii, Z. bisporur, Z lentus and Z rouxii, it is essential to firstly, be able to detect their presence in a food or beverage, and secondly, be able to accuratelyidentify them preferably to the species level. Accurate identification of a food-home yeast can, in many instances, help to establish whether or not the contaminating species represents a spoilage threat to the food product in which it is found. By comparing the physiological characteristics of a yeast contaminant to those of a particular food or beverage, food microbiologistscan often determine the spoilage potential ofthe yeast. For example, if the contaminant is identified as being a highly fermentative yeast species (e. g., S. cerevisiae, T.delbrueckiior Z bailii), product spoilagebecomes a real possibility, if there are fermentable sugars present that the yeast can utilize. Furthermore, knowledge of the yeasts most commonly associated with a particular food product, can also provide insights into the best means of eradicatingthem from the food or beverage (e. g., through addition of weak acid preservatives, or their elimination from raw materials), reducing the likelihood of future incidences of product contamination andor spoilage. Standard methods routinely used to detect for the presence of food-borne yeasts are generally based upon spread plating (see Chapter 2). Typical plating media used include Malt Extract Agar (MEA) and Tryptone Glucose Yeast extract agar (TGY), on to which the food product is either spread directly, or indirectly, having first been homogenised with a suitable aqueous diluent (e. g., 0.1 % peptone [19, 561). If however, the product being tested has a high sugar content (e. g., sugar syrups), then it is often better to use a diluent that has sucrose added to it (to a final concennation of 10 % or higher [ 191). Use of buffered diluents in such circumstances, will often help reduce the death and loss of yeast cells from osmotic shock (as well as aid in the recovery of sub-lethally injured cells), and thus aid in the detection of osmotolerant species such as Z. rouxii. Similarly, diluents containing added salt are recommended for the isolation of yeasts from high salt foods [ 191, Once inoculated, spread plates are typically incubated at either 25 "C or 30 "C, and checked daily for signs of yeast growth (i. e., appearance of discrete colonies). In the case of the Zygosaccharomyces spp. (e. g., Z. bailii), HOCKING [23] found that incubating agar plates at 30 "C, rather than at 25 "C, reduced the time needed to detect the presence of these yeast species, as they grew quicker at the elevated temperature. For preservative-resistant species such as Z. bailii (and indeed other such species e. g., Pichia membranifaciens and Schizosaccharomycespombe),two media routinely used to selectively screen (also referred to as 'target isolation' [ 191) for their presence in products are 180

Detrimental aspects of Zyp~cchammycao

acidified MEA (MAA [i. e., Malt Acetic Agar]) and acidified TGY (TGYA) [56]. In both cases, the basal medium (MEA or TGY) is supplemented with 0.5 % acetic acid. One % acetic acid is used as a key diagnostic test for differentiating Z bailii and Z. bispoms from other Zygosaccharomyces species [6]. As well as these two media, Erickson [16] also developed a medium, ZBM (i. e., Zygosaccharomyces bailii medium), to use for the specific detection of Z. bailii. Like both MAA and TGYA, ZBM medium contains 0.5 % acetic acid, but is additionally supplementedwith 2.5 % NaCl and 0.01 % potassium sorbate. While this medium appears highly selective for detecting the presence of Z bailii in food products, results from a study by HOCKING [23] indicated that this medium might fail to detect the presence of other preservativeresistant species (e. g., Schizosacchuromycespombe).While such yeasts are not as preservative resistant as Z. bailii, they still nevertheless pose a significant spoilage threat. For the detection and isolation of osmotolerant species such as Z. rouxii, typical plating media used generally contain W O % glucose (e. g., MYSOG, i. e., Malt Yeast Extract 50 % Glucose Agar) which lowers the a,of the medium and provides an optimum osmotic pressure to specifically select for these yeasts. However, if initially present in low numbers, yeasts may go undetected in a food or beverage when spread plating methods are used. If the contaminating species is Z. bailii, such an oversight can prove both damaging and costly. Recently, FTm and HOCKING [56] reported that an inoculum of only 5 viable cells of a preservative-adaptedZ bailii strain was sufficient to cause spoilage of a canned carbonated soft drink. Indeed, the authors went on to caution that, in their opinion, presence of even a single healthy cell of Z. bailii would, given sufficient time, lead IO product spoilage, confuming the earlier opinions of VAN ESCH[17] and DAVENPORT [lo], In such circumstances,two techniquesroutinely used (prior to spread plating) to help monitor products for the presence of yeasts in low cell numbers are membrane filtration and inclusion of an enrichment step [56]. Membrane filtration (e. g., hydrophobic grid membrane filtration) is particularly suited for detection of low cell numbers in beverages (i. e., 1 ceW100mls). This technique physically separates viable yeast cells from product ingredients, which may affect yeast growth, and so can improve overall levels of detection. For the reasons given earlier in this section, it is essential that once a yeast is detected in a product, it is reliably and accurately identified, ideally to the species level. Traditionally, this has been achieved on the basis of morphological,physiological and biochemical characterization [6,89]. Typically, such identification requires the use of between 50 and 100 diagnostic tests to reliably identify most yeasts to species level, and routinely takes 1 to 3 weeks to obtain a result (i. e., a species identification). Such a method is both labour intensive and time consuming, and interpretation of resulting data can require considerable expertiSe. Furthermore, accurate identification, to species level, can sometimes be hampered by ambiguous test results due to strain variability [6]. This latter problem can make differentiating between some species difficult, particularly those possessing similar overall physiologies. For example, amongst the Zygosacchuromyces, isolates of Z. bailii and Z bisporus can only 181

Detrimental aspects of Zygxwfccharomyces reliably be differentiated from one another on the basis of their differing responses to a single growth test, namely trehalose assimilation (of the two species, only Z. bailii is capable of assimilating this disaccharide sugar [6,38]). Nevertheless, for practical purposes, several manual and automated yeast identification systems (e. g., API 20C system [BioM6rieux], API ID 32C strips [BioM6rieux],Biolog Microstation system [Biolog Inc.]) have been developed based on this phenotypic approach and are routinely used in many food microbiology laboratories. The degree of success with such techniques varies widely, depending on the skill and experience of the individuals concerned (see also Chapter 5). However, in an effort to improve the overall accuracy and reliability of yeast identification, attention has, in recent years, shifted to the use of DNA-based technologies, such as the polymerase chain reaction (PCR) [57], for the development of alternative methods of identification. PCR-mediatedmethods currently in use for identifying and typing yeasts include DNA sequence analysis of the small- and large-subunit ribosomal RNA encoding genes (i. e., 18s rDNA and 26s rDNA respectively [29,40,41]), random amplified polymorphic DNA (RAPD) analysis [3, 861, and microsatellite PCR fingerprinting [5].

To date, ribosomal DNA (rDNA) sequence analysis has proved by far the most useful method for identifying and characterising yeasts to the species level. This method has gained in popularity since the advent of PCR [57] and the subsequent development of protocols for directly sequencing PCR-amplified DNA products. Development of such protocols has made the amplification and sequencing of yeast ribosomal DNA, either from genomic DNA or directly from individual colonies [29], extremely straightforward. Due to the fact that different regions of the ribosomal DNA display differing levels of sequence divergence has meant that these genes (i. e., 18s and 26s rDNA) and their associated spacer regions (i. e., ITS 1 and ITS2) have proved extremely useful for differentiating between yeasts at a variety of different taxonomic levels (e. g., genus, species and subspecies levels) [18,29, 30.411. With regard to the Zygosaccharomyces species, ribosomal DNA sequence analysis has proved a powerful method for identifying these yeasts [29, 30, 42, 641, and particularly those species difficult to discriminate between using conventional physiological-based approaches (e. g., Z bailii and Z. bisporus; Z. mellis and Z. rouxii [38]). Furthermore, such sequences have also proved useful in the identification of new Zygosaccharomyces species. As discussed in section 6.3.3, Steels and co-workers [a] used 18s rDNA sequences to establish the fact that a number of spoilage-associatedZygosaccharomyces isolates, originally characterised as physiologically atypical Z bailii strains, were in fact representatives of a quite separate and phylogenetically distinct species, which the authors named Z. fentus. More recently, KLTR?ZMAN and colleagues [42], described another new food-associated Zygosaccharomyces, Z. kombuchaenris (isolated from kombucha tea), based on sequence analysis of the variable D1 and D2 domains of the 26s rDNA. In comparison to the 18s rDNA, the DUD2 region of the 26s rDNA displays far more sequence divergence, to the extent that it can be used to differentiate between the vast majority of known yeast species, both ascomycetous and basidiomycetous [18,41].

182

Detrimental aspects of ZygosacchBromyces

Besides their importance in yeast identification, rDNA sequences have also proved useful for studying yeast evolution, and assessing the genealogical relationships (i. e., phylogeny) between different yeast species and genera. In a number of studies, 18S rDNA, 26S rDNA DIID2 and ITS sequences have all been used to investigate the genealogical relationships between species within the genus Zygosaccharomyces [29, 30], and between the Zygosaccharomyces species and those of other related genera [30, 32,41,42, 64]. Results from these studies have demonstrated that the genus Zygosaccharomyces, as currently described [6, 39], is in fact genealogically intermixed with species from other genera (i. e., KZuyveromyces, Saccharomyces and Torulasporai. However despite this phylogenetic intermixing, Z baiZii, Z bisporus and Z rouxii, the three members of the genus most noted for their ability to cause spoilage, are nevertheless genealogically closely related to one another (Fig. 6.32). Indeed these three species, along with Z kombuchaensis [42], Z Zentus [64] and the recently re-established Z mellis [38], form a distinct species group, which is quite separate from other Zygosaccharomyces and non-Zygosaccharomyces yeasts [32, 41, 64]. In their recent description of Z kombuchaensis, KURTZMAN and co-workers [42] refer to this group of species as the Zygosaccharomyces sensu stricto. For identification purposes, ribosomal DNA sequencing is an extremely useful (and accurate) method for identifying and characterizing unknown yeast isolates, as well as differentiating between different yeast species [40, 41]. Generally this is more than adequate for most purposes. However, there may be occasions, particularly if a product is contaminated with more than one yeast strain (either of the same species or of different species), for the need to use alternative typing methods with the ability to resolve below the species level. Two such typing methods that appear to offer this level of (taxonomic) resolution are random amplified polymorphic DNA (RAPD) analysis and microsatellite PCR fingerprinting. In RAPD analysis, DNA fingerprints are generated by the amplification of random DNA fragments by low stringency PCR, using a single small (typically lO-base) oligonucleotide primer of arbitrary nucleotide sequence, and analyzed by subsequent gel electrophoresis [86]. Microsatellite PCR fingerprinting is a related technique, which uses oligonucleotide primers of defined sequence (e. g., [GTGls and [GACls) to amplify simple repetitive DNA sequences and generate a DNA fingerprint pattern. Although similar in principle, the two molecular typing methods differ in the fact that RAPD analysis relies on using a primer annealing temperature that is much lower than that typically used for microsatellite PCR fingerprinting (i. e., 37°C as opposed to 55 "C) [81]. Consequently, problems can arise regarding the reproducibility of DNA fingerprint patterns generated by RAPD analysis, particularly between different typing laboratories. Nevertheless, both PCR-based techniques are quick and easy to execute, require only small amounts of template DNA (not necessarily of high purity), and can be used for screening/typing large numbers of yeast isolates (for further details see Chapter 4). To date, the two typing methods have been tested on a variety of different yeasts, including a number of spoilage-associated species (e. g., S. cerevisiae, Z bailii, Z bisporus and Z rouxiii and found useful for both species and strain discrimination [3,4,5,47]. In a recent

183

Detrimentalaspects of Zygosaccharomyces

study, B U I R A S COUTOet al. [S] compared the use of microsatellite PCR fingerprinting with that of a standard API identification system for typing 126 yeast strains recovered, over a 14-monthperiod, from a mayonnaise and salad dressing production facility. Results from the study showed that microsatellite PCR fingerprinting was a far superior method for identifying all the yeast species recovered during the survey. Indeed, the authors noted that the M I system failed to distinguish between the spoilage species Z. builii and Z bispoms, and could only identify isolates of these two species to the genus level (i. e., as Zygosaccharomyces spp.). In contrast, the microsatellite PCR fingerprinting not only distinguished between these two species, but also between individual strain types as well. In fact, the level of discrimination observed at the sub-species level, led the authors of the study to conclude that this typing method could prove useful for tracking individual spoilageisolates and identifying their source of origin.

6.5

Quality control

Until recently, the main method of quality control (QC) used by the food and beverage indusmes was that of end-product testing. As the name suggests, this method involves the random sampling of the final product, and determining the total number of viable microbes (referred to as the total viable cell count) present in each sample. Typically for yeasts, this is achieved by plating out sample aliquots onto a suitable detection medium (e. g., MEA or TGY agars), incubating the plates at a set temperature (e. g., 25 or 30 "C), and counting the number of yeast colonies that develop after a specific period of time (e. g., 2 to 5 days). However, as a means of assessing the microbial stability of a food or beverage, total viable cell counts are of limited use. For instance, detection of a high number of yeast cells in a product does not necessarily indicate that spoilage is imminent. In the case of beverages of low nutrient content (e. g. wine), the vast majority of yeast contaminants typically present at the time of bottling will fail to survive for more than a few days, and so spoilage of such beverages is unlikely to arise [74]. Likewise, detection of low numbers of yeasts in a product does not guarantee that it will be safe from spoilage. This is particularly true in the case of Z. bailii, a yeast species more than capable of causing soft drink spoilage from an initial inoculum of only one viable cell per container [lo, 17, 561. An alternative approach to end-product testing as a method of quality control, is the use of

quality management systems, such as the Hazard Analysis Critical Control Point (HACCP) system 1451. The aim of the HACCP system is to identify and control the (microbial) hazards associated with the manufacture of a food or beverage, removing the need to test the end product. Hazard analysis consists of analyzing all the procedures and raw ingredients used in the production of a foodheverage. The analysis identifies the critical control points, such as locations, processing equipment or raw ingredients, where hazards (i. e., introduction of microbial contamination) are most likely to occur. Once identified, these points can then be monitored on a regular basis as part of the overall quality control programme, and systems put in place to minimise the risk of microbial infection (e. g., pasteurization of raw 184

Detrimental aspects of ZLgOsacchammyces

ingredients). Recent EC legislation necessitates identification of hazards using the principles of HACCP for all producers of food and drink [l].

6.6

Future prospects and conclusions

At present, the vast majority of yeast spoilage of foods and beverages that have been processed and packaged according to normal standards of good manufacturingpractice can be attributed to a limited number of species [56]. Amongst this list of species (Table 6.1-l), Z builii is without doubt the most problematic spoilage yeast currently encountered by the food and beverage industries, due to its exceptional resistance to preservatives. However, with continual demand for the developmentof new products, the possibility exists for other yeast species to emerge as spoilage threats, some of which may not have been previously encountered or characterized, such as Z. lentus [64]. One potential source for introducing new yeasts (both spoilage and non-spoilage species) into a product or processing facility is through the use of novel ingredients, such as tropical fruits in the manufacture of soft drinks. Furthennore, as well as being potential sources of yeast infection, addition of such ingredients to a product may also alter its overall chemical composition, thereby rendering it susceptible to spoilage by other commonly encountered species. Likewise, changes in preservation strategies may also bring about an increase in product spoilage. Growing consumer concern regarding the presence of chemical residues (e. g., pesticides) in foods and beverages may, in time, lead to a widespread reduction in the levels of chemical preservatives added to foods and beverages. This in turn,may result in an increase in the incidences of spoilage, particularly by yeasts of moderate preservative resistance, such as .'7 delbrueckii and 2.microellipsoides,two species widely distributed in soft drinks factories. At present, the levels of preservative added are sufficient to prevent these species from causing significant product spoilage. However, if in the future preservative levels are reduced, yeasts such as T.delbrueckii and 2.microellipsoides could be encountered much more frequently as spoilage agents. Reliable and effective methods of detection and identification are therefore key to safeguarding foods and beverages from spoilage by yeasts and other microbes, such as bacteria and moulds. Indeed, recent advances in DNA-based technologies, are undoubtedly paving the way towards a more accurate and robust means of identifying yeasts to the species level. 411, have already proved useful in the Methods such as ribosomal DNA sequencing [a, identification of many new yeasts, including two new species of Zygosaccharomyces [42, 641, one of which (2 fentus)has been shown to possess significant spoilage potential [65]. With the amount of yeast sequence data ever increasing, and now including the complete genome sequences for 5'. cerevisiue [20] and Sch. pombe [87], as well as partial genome sequences for a further 13 species [61], there is real scope for the design of species-specific detection systems. While still in their infancy, PCR-based methods have already been developed for detecting the presence of a number of spoilage-associatedyeasts, (e. g., Z len-

185

Detrimental aspects of Zygosaccharomyces

tus [31]; S. cerevisiae, Z bailii and Z. bisporus [52}). Furthermore, with the introduction of PeR-based typing techniques (e. g., microsatellite PeR fingerprinting [81] and RAPD analysis [86]), there is also now the means available for differentiating between individual spoilage strains of either the same or different species, and identifying their sources of origin [5]. With adaptation and development, such methods clearly lend themselves for use in food microbiology laboratories, and inclusion in quality assurance (e. g., RACCP) and quality control programmes as effective microbial monitoring systems.

6.7

References

II]

ANON.: EC Directive 93f94lEEC on Hygiene of Food Stuffs. European Council Official Journal No.L 175/1 (19 mJuly 1993).

[2]

ANON.: EC Directive 95121EC, Food additives other than colours and sweeteners. European Council Official Journal No L. 61 (18 mMarch 1995).

[3]

BAIHRAS COlITO, M.M.; VAN DER VOSSEN, J.M.B.M.; HOFSTRA, H.; HUIS IN'T VEl.[), J.H.J.: RAPD analysis: a rapid technique for differentiation of spoilage yeasts. Int. J. Fd Microbiol. 24 (1994) 249-260.

[4]

BALEIRAS Como, M.M.; voon.s, J.rW.E.; HOFSTRA, H.; HlJIS IN'T vm,o, J.H.J.; VAN DER VOSSEN, J.M.B.M.: Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts. J. Appl. Bacteriol. 79 (1995), 525--535.

[5]

BAlBIRAS Cotrro, M.M.; HARTOO, B.J.; HlJIS IN'T VELD,J.H.J.; HOFSTRA, H.; VANDERVOSSEN, J.M.B.M.: Identification of spoilage yeasts in a food-production chain by microsatellite polymerase chain reaction fingerprinting. Fd Microbiol. 13 (1996), 59-67. BARNETI', J.A.; PAYNE, R.W.; YARROW, D.: Yeasts: characteristics and identification, 3rd ed.

[6]

Cambridge, U.K.: Cambridge University Press (2000).

[7]

COlI', M.B.: The effect of weak acids and pH on Zygosaccharomyces bailii. Ph.D. thesis: University of East Anglia, Norwich, U.K. (1987).

[8]

COLE, M.B.; KEENAN M.H.J.: Effects of weak acids and external pH on the intracellular pH of Zygosaccharomyces bailii, and its implications in weak acid resistance. Yeast 3 (1987) 23-32.

[9]

CONWAY, E.J.; DOWNEY, M.: An outer metabolic region of the yeast cell. Biocbem. J. 47 (1950) 347-355.

[10]

DAVENPORT, R. R.: Forensic microbiology for the soft drinks business. Soft Drinks Management International April (1996) 34-35.

[II]

DAVENPORT, R.R.: Forensic microbiology II, case book investigations. Soft Drinks International April (1997) 26-30.

[12]

DAVENPORT, R.R.: Microbiology of soft drinks. In: Chemistry and technology of soft drinks and fruit juices (edited by Ashurst P.R.). Sheffield, UK: Sheffield Academic Press & CRC Press: Boca Raton (1998) 197-216.

[13]

Do CARMO-SOUSA, L.: Distribution of yeasts in nature. In: The Yeasts, Vol. I (edited by Rose, A.H.: Harrison J.S.). London, U.K.: Academic Press (1969) 79-105.

186

Detrimental aspects of ZygoBIJccharomyces [14]

DRAGON!, L; COM!,G.: Prensenza di muffe e lieviti in succhi di frutta prodotti industrialmente. Ind. Bevande 14 (1985) 599-606.

[l5]

EMMERICH, W.; RADLER P.: The anaerobic metabolism of glucose and fructose by Saccharomyces bailii. J. Gen. Microbiol. 129 (1983) 3311-3318.

[16]

ERICKSON, J.P.: Hydrophobic membrane filtration method for the selective recovery and differentiation of Zygosaccharomyce.f bailii in acidified ingredients. J. Fd Protect. 56 (1993) 234-238.

[17]

VAN EsCH, F.: Yeasts in soft drinks and fiuitjuice concentrates. De Ware (n) Chemicus 17 (1987) 20-31.

[18]

FELL, J.w.; BOEKHOUT, T.; FONSECA, A; SCORZIITTI, G.; STATZELL-TALLMAN, A: Biodiversity and systematics ofbasidiomycetous yeasts as determined by large-subunit rDNA D IID2 domain sequence analysis. Int. J. Syst. Evol. Microbiol. 50 (2000) 1351-1371.

[19]

FLEET,G.: Spoilage yeasts. Crit. Rev. Biotechnol. 12 (1992) 1--44.

[20]

GOFFEAU, A.; BARREL, B.G.; BUSSEY, H.; DAVIS,R.W.; DUJON, B.; FELDMANN, H.; GAUBERT, F.; HOHEISEL, J.D.; JACQ, C.; JOHNSTON, M.; LoUIS, E.1.; MEWES, H.W.; MURAKAMI, Y.; PIDIJPPSEN, P.; TEITELIN, H.; OUVER, S.G.: Life with 6000 genes. Science 274 (1996) 543567.

[21]

GRINBAIJM, A; AsHKENAZI, I.; TREISTER, G.; GoLDSCHMlED-REoINEN, A; BLOCK, C.S.: Exploding bottles: eye injury due to yeast fermentation of an uncarbonated soft drink:. Br. J. OphthalmoJ. 78 (1994) 883.

[22]

HENRlQUES, M.; QUlNTAS, C.; UJUREIRo-DIAS, M.C.: Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism. Microbiology 143 (1997) 1877-1883.

[23]

HOCKING, A. D.: Media for preservative resistant yeasts: a collaborative study. Int. J. Fd MicrobioI. 29 (1996) 167-175.

[24]

HOLYOAK, C.D.; STRATFORD, M.; McMUUJN, Z.; COLE, M.B.; CRIMMINS, K.; BROWN, A.1.; COOTE,P.1.: Activity of the plasma membrane W-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative, sorbic acid. Appl. Envir. Microbiol. 62 (1996) 3158-3164.

[25]

HOLYOAK, C.D.; BRACEY, D.; PiPER, P. W.; KUCHLER, K.; COOTE, P.1.: The Saccharomyces cerevisiae weak-acid inducible ABC transporter Pdr12 transports fluorescein and preservative

anions from the cytosol by an energy-dependant mechanism. I. Bacteriol. 181 (1999) 46444652. [26]

INGRAM, M.: Studies on benzoate-resistant yeasts. Acta Microbiol. 7 (1960) 95-105.

[27]

INGRAM, M.; OTTAWAY, F.J.H.; COPPOCK, I.B.M.: The preservative action of acidic substances in food. Chern. Ind. (London) 75 (1956) 1154-1163.

[28]

ISON,R.w.; GIJ'ITERlDOE, C S.: Determination of the carbonation tolerance of yeasts. Lett. Appl. Microbiol. 5 (1987) 11-13.

[29]

JAMES, SA; COLLINS, M.D.; ROBERTS, LN.: Genetic interrelationship among species of the genus Zygosaccharomyces as revealed by small-subunit rRNA gene sequences. Yeast 10 (1994) 871-881.

[30]

lAMES, S.A.; COLlJNS,M.D., ROBERTS, I.N.: Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora. Int. 1. Syst. BacterioL 46 (1996) 189-194.

187

Detrimental aspects of Zygosacchsromyces [31]

JAMES, S.A; COLLINS, M.D.; ROBERTS, LN.: Genetic analysis of food spoilage yeasts. In: Methods in Biotechnology, Vol. 14, Food Microbiology Protocols (edited by Spencer, J.F. T.: Ragout de Spencer, A.L.). Totowa, New Jersey, U.S.A.: Humana Press (2001) 37-51.

[32]

JAMES, S.A.; CAl, J.; ROBERTS, LN.; COlLINS, M.D.: A phylogenetic analysis of the genus S(JL'charomyces based on 18S rRNA gene sequences: description of Saccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. nov. Int. J. Syst. Bacteriol. 47 (1997) 45J....460.

[33]

JERMINI, M.F.G.; GElGES, 0.; SCHMIDT-LoRENZ, W.: Detection, isolation and identification of osmotolerant yeasts from high-sugar products. J. Fd Protect. SO (1987) 468--472.

[34]

KABARA, 1.1.; EKLUND, T.: Organic acids and esters. In: Food Preservatives (edited by Russell, N.J.: Gould, G.W.). Glasgow and London, U.K.: Blackie Academic & Professional (1991) 457I.

[35]

KATO, N.: Antimicrobial activity of fatty acids and their esters against a film-forming yeast in soy sauce. J. Fd Safety 3 (1981) 121-126.

[36]

KOTYK, A: Uptake of 2,4- dinitrophenol by the yeast cell. Folia Microbiol. (Prague) 7 (1962) 10'}-1l4.

[37]

KREBS, H.A; WIGGINS, D.; STUBS, M.; SOI8, A.; BEIX)YA,F.: Studies on the mechanism of the antifungal action of benzoate. Biocbem. J. 214 (1983) 657--{i63.

[38]

KURTZMAN, C.P.: DNA relatedness among species of the genus Zygosaccharomyces. Yeast 6 (1990) 213--219.

[39]

KURTZMAN, c.P.: Zygosaccharomyces Barker. In: The yeasts: a taxonomic study, 4 th ed. (edited by Kurtzman, C. P.: Fell, J. W.). Amsterdam, the Netherlands: Elsevier (1998) 424-432.

[40j

KURT7MAN, C.P.; BLANZ, P.A.: Ribosomal RNAJDNA sequence comparisons for assessing phylogenetic relationships. In: The yeasts: a taxonomic study, 4 th ed. (edited by Kurtzman, C.P.: Fell, J.w.). Amsterdam, the Netherlands: Elsevier (1998) 69-74.

[41]

KURTZMAN, C.P.; ROBNlnT, C.J.: Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73 (1998) 331-371.

[42]

KURTZMAN, C.P.; ROBNETT, C.J.; BASEHOAR-POWERS, E.: Zygosaccharomyces kombuchaensis, a new ascosporogenous yeast from 'Kombucha tea'. FEMS Yeast Research 1 (2001) 133-138.

[43]

LACHANCE, M-A.; STARMER, W.T.: Ecology and yeasts. In: The yeasts: a taxonomic study, 4 th ed. (edited by Kurtzman, c.P.: Fell, J.w.). Amsterdam, the Netherlands: Elsevier (1998) 21-30.

[44]

LEGAN, J.D.; VOYSEY, P.A. : Yeast spoilage of bakery products and ingredients. J. Appl. Bacteriol. 70 (1991) 361-371.

[45]

MAYES, T.: HACCP training. Fd ControlS (1994) 190-195.

[46]

MOLLAPOIJR, M.; PIPER, P.W.: The ZbYME2 gene from the food spoilage yeast Zygosaccharomyces bailii confers not just YME2 functions in Saccharomyces cerevisiae, but also capacity for catabolism of sorbate and benzoate, two major weak organic acid preservatives. Mol. Microbiol. 42 (2001) 919-930.

[47]

MOLNAR, 0.; MESSNER, R.; PRl1HNGER, H.; STAHL, U.; SLAVIKOVA, E.: Genotypic identification of Saccharomyces species using random amplified polymorphic DNA analysis. Syst. Appl. Microbiol. 18 (1995) 136-145.

188

Detrimental aspectsof ZygoBBcchBromyces [48]

NEAL, A.L.; WEINSTOCK, J.O.; Bacteriol, 90 (1965) 126--13l.

[49]

NEVES, L.; PAMPUU1A, ME; UmREIRo-DlAS, M.C.: Resistance of food spoilage yeasts to sorbic acid. Lett. AppL MicrobioL 19 (1996) 8--11.

[50]

PARK, H.; BAKAlJNSKY, A.T.: SSUI mediates sulphite efflux in Saccharomyces cerevisiae. Yeast 16 (2000) 881-888.

[51]

PEARCE, A.K.; BOOTH, LR.; BROWN, A.J.P,: Genetic manipulation of6-phosphofructo-1-kinase and fructose 2,6-bisphosphate levels affects the extent to which benzoic acid inhibits growth of Saccharomyces cerevisiae. Microbiology 147 (2001) 403-410.

[52]

PEARSON, B.M.; MCKEE, RA.: Rapid identification of Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii. Int. J. Fd Microbiol. 16 (1992) 6'J--67.

[53]

PILKINGTON, B.J.; ROSE, A.H.: Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite. J. Gen. MicrobioL 134 (1988) 2923-2830.

[54]

PIPER, P.W.; ORTIZ-CALDERON, C.; HOLYOAK, C.D.; COOTE, P.J.; AND COLE, M.B.: Hsp30, the integral plasma membrane heat shock protein of Saccharomyces cerevisiae, is a stress-inducible regulator of plasma membrane ATPase. Cell Stress Chaparones 2 (1997) 12-24,

[55]

PIPER, PW.; MAIlE, Y.; THOMPSON, S.; PANDJAlTAN, R; HOLYOAK, C.D.; EGNER, R; MUHLBAUER, M,; COOTE, P.J.; KUCHLER, K.: The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. EMBO J. 17 (1998) 4257-4265.

[56]

PrIT,

LAMPEN,

J.O.: Mechanisms of fatty acid toxicity for yeast. J.

J.L; HOCKING, A.D.: Fungi and food spoilage, 2nd ed. London, U.K.: Blackie Academic

& Professional (1997).

[57]

SAIKI, R.K.; GELFAND, D.H.; STOFFEL, S.; SCHARF, S.J.; Htouctn, R; HORN, G.T.; MtJUlS, K.B.; EHRliCH, H.A.: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 (1988) 487-491.

[58]

SAlTOU, N.; NEl,M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees. MoL BioL Evol. 4 (1987) 406-425,

[59]

SAND, F.E,M.J.; VAN GRlNSVEN, A.M.: Comparison between the yeast flora of Middle Eastern and Western European soft drinks, Antonie van Leeuwenhoek 42 (1976) 523-532.

[60]

SAND, F.E,M.J.; VAN DEN BROER, W.C.M.; VAN GRINSVEN, A.M.: Yeasts isolated from concentrated orange juice. Proc. 5th Int. Spec. Symp. Yeasts (1977) 121-122.

[61]

SOUClIIT, J.-L.; A1GLE, M.; ARTIGUENAVE, F.; BLANDIN, G,; BOLarEN-FtlKlffiARA, M.; et al.: oenolevures: genomic exploration of the hemiascomyeetOllS yeasts.FEBS Letters 487 (200D) 1-149,

[62]

SOUSA, M.J,; MIRANDA, L.; CORTE-REAl., M.; LEAO, C.: Transport of acetic acid in ZygO.faccharomyces bailii - effects of ethanol and their implications on the resistance of the yeast to acidic environments. Appl. Envir. Microbiol. 62 (1996) 3152-3157.

[63]

SOUSA, M.J.; RODRIGUES, F,; CORTE-REAL, M.; LEAo, C.: Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii. Microbiology 144 (1998) 665-670.

c.r., COUJNS, M.D.; ROBERTS, LN.; STRATFORD, M.; JAMES, SA: Zygosaccharomyces lentus sp. nov., a new member of the yeast genus Zygosaccharomyces Barker.

[64J STEELS, H.; BOND,

Int. J. Syst. Bacteriol. 49 (1999) 319-327.

189

Detrimental aspects of Zygosaccharomyces [65)

STEELS, H.; JAMES, S.A; ROBERTS, LN.; STRA'IHlRD, M.: Zygosaccharomyces lenius: asignificant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature. J. Appl. Microbiol. 87 (1999) 520-527.

[66)

STEEI.~, H.; JAMES, S A.; ROBERTS, LN.; STRA1FORD, M.: Sorbic acid resistance: the inoculum effect. Yeast 16 (2000) 1173-1183.

[67]

STRA'!'FORD, M.: Sulphite metabolism and toxicity in Saccharomyces cerevisiae and Saccharomycodes ludwigii. Bath, UK: Ph.D thesis, University of Bath (1983).

[68)

STRAll'(lRD, M.; ANSLOW P.A.: Evidence that sorbic acid does not inhibit yeast as a classic "weak-acid preservative". Lett. AppL Microbiol. 27 (1998) 203-206.

[69]

STRAn'oRD, M.; ROSE, A. H.: Transport of sulphur dioxide by Saccharomyces cerevisiae. 1. Gen. MicrobioL 132 (1986) 1--6.

[70]

STRA'J1IORD, M.; MORGAN, P.; ROSE, A.H.: Sulphur dioxide resistance in Saccharomyces cerevisiae and Saccharomycodes ludwigii. J. Gen. MicrobioL 133 (1987) 2173-2179.

[71]

STRAn'ORD, M.; HOFMAN, P.D; COlli, M.B.: Fruit juices, fruit drinks and soft drinks. In: The Microbiological Safety and Quality of Food (edited by Lund, B. M.; Baird- Parker, T. c.; Gould, G. W.). Gaithersburg, Maryland, U.S.A.: Aspen Publishers (2000) 836--869.

[72]

SU7ZI, G.; ROMANO, P.: Flocculazione in Zygosaccharomyces. Ind. Bevande 19 (1990) 306308.

[73]

TF.NRF.IRO, S.; ROSA, P.c.; VIEGAS, C.A; SA-CORREIA, L: Expression of the AZRI gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae. Yeast 16 (2000) 1469-1481

[74]

THOMAS, D.S.: Yeasts as spoilage organisms in beverages. In: The Yeasts: Yeast technology, Vol. 5, 2nd ed. (edited by Rose, A H.: Harrison, J. S.). London, UK: Academic Press (1993) 517-561

[75]

THOMAS, D.S.; DAVENPORT, RR: Zygosaccharomyces bailii - a profile of characteristics and spoilage activities. Fd MicrobioL 2 (1985) 157-169.

[76]

TILBURY, R.H.: Xerotolerant yeasts at high sugar concentrations. In: Microbial growth and survival in extremes of environment (edited by Gould, G. W.; Corry, J. E. L.). London, U.K: Academic Press (1980) 103-128.

[77]

TODD, E.C: Foodborne disease in Canada - a 5-year summary. J. Fd Protect. 46 (1983) 650657.

[78]

TUDOR, E.A; BOARD, R.G.: Food-spoilage yeasts. In: The Yeasts: Yeast technology, VoL 5, 2nd ed. (edited by Rose, AH.: Harrison, J. S.). London, UK: Academic Press (1993) 43~516.

[79]

VAUEJO, CG.; SERRANO, R: Physiology of mutants with reduced expression of plasma membrane H+-ATPase. Yeast 5 (1987) 307-319.

[80]

VOU,EKOVA, A.; MALIK, F.; VOU,EK, V.; LINC7.ENYIOVA, K: Characterization of yeasts isolated from red wine surface film. Folia MicrobioL 41 (1996) 347-352.

[81]

VAN DER VOSSEN, J.M.B.M.; HOFSTRA, H.: DNA based typing, identification and detection systems for food spoilage microorganisms: development and implementation. Int. 1. Fd Microbiol. 3 (1996) 3~9.

[82)

WARTH, A.D.: Mechanism of resistance of Saccharomyces bailii to benzoic, sorbic and other weak acids used as food preservatives. J. Appl. Bacteriol. 43 (1977) 215-230.

190

Detrimental aspects of Zygosaccharomycetl [83]

WARTII, A.D.: Effect of benzoic acid on growth yield of yeasts differing in resistance to preservatives. Appl. Envir. Microbiol. 54 (1988) 2091-2095.

[84]

WARTII, A.D.: Relationships between the resistance of yeasts to acetic, propionic and benzoic acids and to methyl paraben and pH. Int. J. Fd Microbiol. 8 (1989) 343-349.

[85]

W ARTII, A.D.: Transport of benzoic acid propionic acids by Zygosacchnromyces bailii. J. Gen. Microbiol.135 (1989) 1383-1390.

[86]

WilllAMS, J.G.K.; KUBEUK, A.E.; LEVAK, KJ.; RAFALSKI, J.A.; TINGEY, S.C.: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acid Res. 18 (1990) 6531"'{)335.

[87]

wooo, V.; Gwn.UAM, R.; RAJANDREAM, M.-A.; LYNE, M.; LYNE, R.; et aI.: The genome sequence of Schizosaccharomyces pombe. Nature 415 (2002) 871-880.

[88]

YARROW, D.: Zygosaccharomyces Barker. In: The yeasts: a taxonomic study, 3 rd ed. (edited by Kreger-van Rij, N. J. W.). Amsterdam, The Netherlands: Elsevier (1984) 449-465.

[89]

YARROW, D.: Methods for the isolation, maintenance and identification of yeasts. In: The yeasts: a taxonomic study, 4 th ed. (edited by Kurtzman, C.P.: Fell, J.W.). Amsterdam, the Netherlands: Elsevier (1998) 77-100.

191