Normal Cell

Driven into cell by H* gradient

Food

Cell membrane :—3rmeable to H

Food

Cell treated with lso-a-acids

Food

Cell treated with lso-a-acids

Food lso-a-acids allow l-T to enter cell

No H* gradient so food cannot be transported and the cell starves

Fig. 8.9 Effect of iso-a-acids on the physiology of sensitive bacterial cells (after Simpson, 1993).

yeasts is diffuse and, for convenience, is traditionally divided into (i) Saccharomyces and (ii) non-Saccharomyces. Irrespective of classification, wild yeast contamination of process and product can be a major cause for concern. This reflects their diversity, difficulty of detection and, for Saccharomyces wild yeast, similarity to pitching yeast. These issues are compounded by the robustness of wild yeast which is usually equivalent (or exceeds) that of primary yeasts. It is salient to note that the panacea of acid washing is not effective with wild yeasts and control must be achieved via attention to hygienic operations. It is worth noting that 'contamination' need not be overt, characterised by strong off flavours and turbidity. More subtle contamination by other brewing strains can perturb process and product. Excellent reviews on wild yeasts in brewing are to be found in Rainbow (1981) and Campbell (1996) in Brewing Microbiology. Other useful but more broad-based reviews are to be found in The Yeasts (Thomas, 1993) and the Handbook of Food Spoilage Yeasts (Deak & Beuchat, 1996).

8.1.3.1 Saccharomyces wild yeast. These days, the Saccharomyces wild yeasts are regarded as more hazardous than the heterogeneous grouping of the non-Saccharomyces wild yeasts. As noted elsewhere (Section 4.1.3), of the 75 yeast genera tested by Visser et al. (1990), only 23% were able to grow anaerobically and, of these, S. cerevisiae stood out as being capable of robust growth in the absence of oxygen. The opportunities during the brewing process and in final package for contamination and growth by facultative anaerobes like the Saccharomyces are clearly more significant than for exclusively aerobic non-Saccharomyces yeasts. Indeed, the opportunity for the later class is diminishing further with the worldwide focus on tightening dissolved oxygen specifications and decline in more aerobic packaging formats such as cask beer in the UK.

Given the taxonomic consolidation described elsewhere (Section 4.1.1), it is no surprise that typical brewery Saccharomyces spoilage yeasts (Wiles, 1953) such as S.

turbidans, S. ellipsoideus, S. willianus and S. diastaticus are now reclassified as S. cerevisiae. Spoilage strains of S. bayanus and S. pastorianus have remained unscathed by taxonomic changes. As noted above, cross contamination of an ale fermentation with a lager strain (i.e. S. pastorianus) can trigger changes in process and product, and, consequently, cannot be dismissed lightly.

Dramatic changes are seen by contamination with diastatic strains of S. cerevisiae (formerly S. diastaticus). First characterised in 1952 (Andrews & Gilliland, 1952), this yeast expresses a glucoamylase and is able to ferment wort oligosaccharides ('dextrins') that are normally unfermentable with brewing strains of S. cerevisiae. Contamination, which can occur in fermenter or downstream, has a major impact on product quality, resulting in beers with (i) an atypically low present gravity, a phenomenon called 'superattenuation' and (ii) a phenolic, medicinal aroma. This later observation is characteristic of diastatic as well as other 'wild' (but non-diastatic) strains of S. cerevisiae (Ryder et al., 1978). Such yeasts contain an active POF gene (Goodey & Tubb, 1982) that results in a phenolic off-flavour through expression of phenolic acid decarboxylase. This enzyme is able to decarboxylate wort phenolic acids such as ferulic and cinnamic acid to, respectively, 4-vinyl guaiacol (clove/spicy aroma) and styrene (medicinal aroma). It is of interest to note that brewing strains which do not express POF contain a non-functional POF gene (Meaden & Taylor, 1991; Daly et al., 1997). Not surprisingly, the expression of glucoamylase and POF underpins laboratory detection of these yeasts (see Section 8.3.3.2). The genetics and physiology of diastatic strains of S. cerevisiae have been subject to widespread study, as the glucoamylase from this yeast was an early candidate for genetic manipulation into brewing strains for the production of 'light' beers (see Section 4.2.4).

Another more threatening class of wild yeasts are the 'killer' yeasts. These pose a 'greater threat than does the presence of other wild yeasts, since they not only compete for substrate but actively kill the indigenous brewing strain' (Hammond & Eckersley, 1984). These yeasts produce exotoxins or 'zymocins' (to which they are immune) that kill susceptible cells belonging to the same species. The Saccharomyces killer yeasts are by far the best understood although other distinctly different yeast systems have been described in Kluyveromyces, Pichia and Williopsis. Excellent reviews are to be found in Young (1987), Magliani et al. (1997) and Walker (1998).

Saccharomyces killer strains have been classified into three main groups - Kl, K2 and K28 - according to the toxins they secrete. All are unusual in that the genetic basis is not chromosomal but is coded for by different cytoplasmically inherited double stranded RNA plasmids. Although different, Kl and K2 toxins both bind to (3-1,6-glucan receptors in the cell wall and then penetrate the cytoplasmic membrane. These events cause the cell to become 'leaky' such that ions are lost and the cell dies. Conversely, the mode of action of K28 involves binding to the a-l,3-mannose residues in the wall mannoprotein which leads to cell cycle arrest through inhibition of DNA synthesis and the non-separation of mother and daughter cells.

The ecology of the killer yeast phenomenon has been studied primarily in yeast communities such as decaying fruits and in slime growth in trees. The occurrence of killer yeasts in brewing has had a mixed press. In a major survey of 964 yeasts (28 genera) in the National Collection of Yeast Cultures (see Section 4.2.3.1), Philliskirk and Young (1975) found 59 killer strains. The vast majority (38) were of the genus

Saccharomyces, of which 27 strains were laboratory haploids, rather than industrial yeasts. No more than four of the killer yeasts were connected with brewing. This is in keeping with the limited number of reports of Saccharomyces killer yeasts in brewery fermentations. The first (Maule & Thomas, 1973) described the occasional colonisation of a production-scale two-stage stirred continuous fermenter of the type described by Bishop (1970) (see Section 5.6.2.2). So effective was the killer yeast that on reaching 2-3% of the total yeast mass, the primary yeast viability fell from greater than 95% to less than 20%. From the perspective of Saccharomyces wild yeast, it is noteworthy that the beer acquired a 'herbal/phenolic' taint, consistent with the expression of POF as described above. This problem was eliminated, as ever, by effective cleaning and sterilisation. Similarly, the other report of killer yeasts in production brewing (Taylor & Kirsop, 1979) described the contaminated beer as having an unpleasant phenolic aroma. Unlike the earlier report, this example of Saccharomyces wild yeast was isolated from a batch fermentation. Not surprisingly, the heavy flocculence of the killer yeast facilitated its transfer between conical fermenters, whereas - in the same brewery - traditional vessels cropped via skimming were not colonised.

The introduction of killer factor into brewing strains was an early candidate in the various 'strain improvement' programmes (see Section 4.2.4) of applied yeast geneticists in the early 1980s (Young, 1981; Hammond & Eckersley, 1984). Simplis-tically, such developments are of value in managing out the threat of contamination by other primary yeasts or wild Saccharomyces strains. The then technical complexity coupled with today's concerns and reticence over genetic modification suggest that contamination is best controlled - as ever - by attention to hygienic practices and CiP.

8.1.3.2 TVora-Saccharomyces wild yeast. The collection of yeasts grouped together as the 'non-Saccharomyces wild yeasts' is notable for its complexity and ambiguity. Indeed, it is perhaps this, together with the frustration of identification, that has hampered the detailed study of the numerous genera of yeasts that are included in the non -Saccharomyces. As noted above, this diverse collective is considered to pose less of a general threat to product quality than the Saccharomyces wild yeast. Simplisti-cally, the non-Saccharomyces wild yeast are less well equipped to spoil beer than the Saccharomyces strains. Indeed, 'head to head' there is no contest! The few comparative reports (Visser et al., 1990; Campbell & Msongo, 1991) show, as would be expected, that the Saccharomyces yeasts are better adapted to the environment of the brewing process and product. As a class, the non-Saccharomyces yeasts grow, at best, poorly under anaerobic conditions and frequently are either unable to ferment sugars or ferment a reduced spectrum of sugars. Despite such a damning comparison, the non -Saccharomyces yeasts found in the process or product includes a wide diversity of genera (see Table 8.3). Of those found in breweries, Candida and Pichia species predominate (Wiles, 1953; Hall, 1971; van der Aa Kuhle & Jespersen, 1998). Although frequently endemic in a brewery or process, in isolation such yeasts are unable to spoil packaged beer in the absence of oxygen. These organisms are typically removed through pasteurisation or sterile filtration. Although they are no threat to product quality, such yeasts are a major irritation inasmuch that they are detected

Table 8.3 Non- Saccharomyces wild yeast.

Genus

Major species

Characteristics*

Beer spoilage

Brettanomyces

B. anomalus B. bruxellensis B. lambicus

Teleomorph (similar morphology, physiology) of Dekkera, oxygen stimulates fermentation, ferment glucose, rarely maltose but not sucrose, produce acetic acid, nitrate reducing

Notable for causing off-flavour in bottle conditioned beers, succeed Saccharomyces in the spontaneous fermentation of wort (Iambic and gueuze)

C. boidinii

C. vini (formally C. mycodema)

Fermentation typically limited to glucose (C. tropicalis can ferment maltose)

Infection limited to the initial 'aerobic' phase of fermentation or unpasteurised draught beers, reports that some strains can grow poorly anaerobically

Cryptococcus

C. laurentii

Unable to ferment but can assimilate a wide range of sugars, some strains produce pigments

Can be found in beer in process or in package, survives but does not spoil

Dekkera

D. bruxellensis D. hansenii

Teleomorph of Brettanomyces forming ascospores, oxygen stimulates fermentation, ferment glucose, sucrose and maltose (strain variable)

Spoils unpasteurised draught beer

Kluyveromyces

K. marxianus

Glucose is fermented vigorously, other wort sugars are variable as is lactose fermentation, thermotolerant (growth 37^13°C)

Spoils soft drinks, fruit juices and high-sugar products, common contaminant of dairy products

Pichia (including Hansenula)

P. anomala P. fermantans P. membranifaciens

Fermentation usually limited to glucose

Infection limited to the initial 'aerobic' phase of fermentation, can spoil unpasteurised draught beer, forms haze and surface films, P. membranifaciens reported to give a sauerkraut flavour

Rhodotorula

R. glutinis R. mucilginosa

Some strains are pigmented red, unable to ferment but can assimilate a wide range of sugars

Water borne, found in pitching yeast, can survive but not spoil beer

Torulaspora

T. delbrueckii

Ferments glucose and variably maltose and sucrose, phenotypically close to Saccharomyces

Pitching yeast contaminant, can spoil unpasteurised draught beer, capable of poor anaerobic growth

Zygosaccharomyces

Z. bailii Z. rouxii

Ferments glucose and variably maltose and sucrose, notably osmophillic

Infamous spoilage organism of soft drinks, fruit juices and high-sugar products

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