Bcg

* The sheer diversity and complexity of cell size and shape together with appearance on plates or in liquid culture undermine the value of any attempt at a generalised description of each genus. The interested reader should consult Kurtzman and Fell (1998) in The Yeasts, A Taxonomic Study for specific details.

through routine microbiological monitoring. Irrespective of their limited threat, such yeasts are a valuable indication of the status of the hygiene of the process. Accordingly, steps should be taken to identify the source of such yeasts so that they can be eradicated from the process and the product. After all, it is important to recognise that the presence of non-threatening contaminant yeasts may be the 'tip of the iceberg' that hides the presence of other, potential beer spoilage organisms.

Some non-Saccharomyces wild yeasts are typically 'niche' contaminants that come into their own under specific conditions. For example, Brettanomyces species which are 'key players' in Belgian beers such as Iambic and gueuze (Van Oevelen et al., 1977) are contaminants of bottle conditioned beers (Gilliland, 1961). Here, these slow growing yeasts are described as producing flavours varyingly described as 'harsh', 'mawkish' and 'old beer flavour' (Gilliland, 1961). Unlike the Saccharomyces, the Brettanomyces are more 'fermentative' under aerobic conditions than anaerobically.

A broader example of niche contaminants is the colonisation of traditional draught 'cask' beers in the UK. Here, it is usual practice for air to be drawn into the container as it is emptied, with the result that any aerobic yeasts that are present can grow and, over time, spoil the beer. The sheer diversity of contaminant wild yeasts found in cask beers was reported in the bad old days of the 1950s when returns 'could be disastrously high on occasions' (Harper et al., 1980). In a survey of various public houses in London and Surrey, Hemmons (1954) analysed 41 samples ex-dispense of 19 different brands. Although Saccharomyces wild yeast predominated, aerobic, film-forming yeasts such as Pichia membranifaciens and Candida mycoderma (now C. vini) were also prevalent. A survey some 26 years later (Harper et al., 1980) found a similar spectrum of wild yeasts in dispensed cask beers. This is not surprising, as the origin of contaminant organisms in cask beers is unlikely to have changed in the 1950s, '80s or, indeed, today! Although contaminant yeasts can already be present in the product in low numbers it is more likely that they are introduced via the air, poor cellar hygiene or 'grow back' from dispense lines. Arguably today, cask beer spoilage is less of an issue with improvements in product hygiene, cellar management, the use of inert gas blankets and reduced time on dispense. A further consideration is the commercial decline in cask beer which has resulted in the 'selection' of those 'who look after the beer' continuing to sell these more demanding products.

Similarly, the microbiology of keg beer in the bar and public house can be of concern. Indeed, it is both ironic and disappointing that keg beer leaves the brewery 'commercially sterile' only to become contaminated to a greater or lesser degree on dispense. Remarkably, given its importance in delivering high-quality product, there have been few published reports that focus on the microbiology of keg beers in the trade. Like cask beer, keg beer is subject to varying degrees of contamination by a variety of bacteria and yeasts. In an extensive survey of over 600 samples of keg beers, Harper et al. (1980) reported the presence of wild Saccharomyces strains together with Hansenula, Pichia, Torulaspora and less commonly Brettanomyces, Debaromyces and Kloeckera. It is noteworthy that Harper et al. (1980) reported that 'in general, levels of both yeasts and bacteria were considerably lower than in the case of cask beer'. Given that keg beers, unlike cask conditioned beers, are pasteurised or sterile filtered this observation is both reassuring and no great surprise.

8.1.4 Sources

Hammond et al. (1999) commented that it 'must be realised that the brewing process is not aseptic and the occasional chance contaminant will often be encountered'. Although for some perhaps a rude awakening, this is a helpful, practical and, frankly, realistic comment. The reality is that packaged beer is, at best, 'commercially sterile'. Although this is more than acceptable in terms of product quality and robustness, it does mean that beer can realistically contain relatively low levels of contaminants. However, in this event the loading is such that they are typically undetectable in routine sampling and testing and, most importantly, do not pose a threat during the shelf-life of the product. Consequently, the focus of brewing microbiology has been 'calibrated' to routinely deliver a realistic low loading of micro-organism such that the shelf-life of the packaged product is not compromised. Accordingly, the process is managed to minimise the threat of significant microbial entry and contamination.

Typically this is achieved by minimising the ingress of organisms (closed vessels), cold storage of beer in process and removal from surfaces by robust cleaning (CiP) operations. Operationally, microbial loading is reduced via process steps such as wort boiling, acid washing, filtration, pasteurisation and/or sterile filtration (see Section 8.2).

8.1.4.1 Biofilms. Biofilms have been described as 'the real enemy of process and product hygiene' in brewing (Quain, 1999). Since the 1970s, it has become increasingly obvious that in the 'real world' micro-organisms attach themselves to surfaces and form quasi 'multicellular' mixed communities. These 'biofilms' or 'biosponge' provide both protection and nutrients for micro-organisms in what are typically hostile unwelcoming environments. Biofilms are everywhere! Classic biofilms include dental plaque, slimes in water pipes and drains and biofouling in cooling towers (cf. Legionnaires' disease) and heat exchangers. The growing realisation that biofilms colonise food preparation surfaces and medical implants and catheters has triggered a step change in the profile and financial support for work in this area. Further fuel has been provided by the report from the Centre for Disease Control and Prevention in the USA that 65% of human bacterial infections involve biofilms. A review of the role of biofilms in human infections has been published by Costerton et al. (1999). Indeed, it is a sobering thought that it has been estimated that 99% of the planet's bacteria live in biofilms (Coghlan, 1996). By contrast, the free flowing 'planktonic' bacteria so typical of laboratory studies represent only a very small fraction of the microbial community. Seemingly, the decision to enter the biofilm mode of growth is determined by the availability of an external carbon source (Pratt & Kolter, 1999). If the concentration is low, the biofilm option is taken up as, presumably, being better for foraging low levels of nutrients. Conversely, the dispersal of unattached cells and sheared fragments of biofilms act as the 'advance party' to colonise other areas in the environment.

Numerous reviews on biofilms have been published (see Keevil et al., 1995; Coghlan, 1996; Costerton et al., 1995; 1999; Stickler, 1999). This interest in biofilms has spawned some rich descriptions that aid the visualisation of these three-dimensional structures. Of particular note was the article by Coghlan (1996) in the weekly UK popular science magazine New Scientist. He reported biofilms as being 'slime cities' resembling 'skyscrapers of ghostly spheres piled one on top of the other' and, most memorably, as looking 'like Manhattan when you fly over it'. Such descriptions infer, quite correctly, that biofilms are organised, arranged structures. Figure 8.10 shows a schematic biofilm consisting of a basal layer 5-10 (im thick covered with pillar and mushroom shaped stacks or towers of microcolonies that rise 100-200 (im above the attached surface. The basal layer consists of a microbially derived extra-polysaccharide (EPS) matrix that aids attachment and provides protection for encapsulated microbes. The chemistry of the EPSs found in biofilms varies greatly - so much so that their stickiness varies from 'velcro' to 'superglue' (Sutherland, 1997). The availability of nutrients, which are transported through channels in the biofilm, determines the density of the 'skyscrapers'. Although an idealised description, biofilms are composed of a complex consortia of microorganisms that provide niche environments for pure and mixed communities. This is key to the success of biofilms, as through pooling metabolic resources, nutrients are ruthlessly utilised. In such an environment, the metabolic by-products of one community are the food of another.

currents ^ E

Bacterial

Surface thick slime

Fig. 8.10 Schematic diagram of a biofilm (after Keevil et al., 1995 and Coghlan, 1996).

To add further complexity and weight to the view that biofilms are 'rudimentary organs' (Keevil et al., 1995), there is persuasive evidence for cell-to-cell communication within these communities. Signal peptide molecules of the acyl homoserine lactone (AHL) family have been shown to accumulate in growing planktonic cultures of over 30 species of Gram-negative organisms. This 'quorum sensing' (Bassler, 1999) enables bacteria to sense population numbers and to coordinate gene expression of the entire population in direct response to the accumulation of AHLs. Although it is thought that quorum sensing provides a selective advantage in regulating the virulence of pathogenic bacteria, such signalling also plays an important role in biofilm development (Heys et al., 1997). Davies et al. (1998) demonstrated that in Pseudomonas aeruginosa, 3-oxododecanoyl homoserine lactone (30Ci2-HSL) is required for biofilm differentiation but not attachment. A mutant unable to secrete this extracellular signal produced thin, undifferentiated biofilms, which were sensitive to dispersion by a weak detergent. The addition of 30Ci2-HSL to the mutant restored the formation of normal, biocide resistant biofilms.

Not surprisingly, there has been much interest - particularly in medicine - in prevention of biofilm attachment to surfaces. A popular route has been to incorporate biocides into the substrate so that they are intrinsically resistant to microbial colonisation (Stickler, 1997). Approaches have included coating the surface with silver, copper and incorporation of antibiotics. Indeed, incorporation of the biocide triclosan into materials has been exploited commercially by inclusion into a wide variety of bathroom products, kitchen utensils and household goods (Quain, 1999). Other approaches include physico-chemical modification of surfaces such that they are more hydrophilic (Stickler, 1997) or the use of'smart' polymers that respond to the environment (Ista et al., 1999). A more recent concept is to interfere with cell-to-cell communication so as to exploit the above observations so that biofilms become more susceptible to removal (Davies et al., 1998).

From our perspective, the real issue is that biofilms are significantly more resistant to removal and death through treatment with detergents and biocides. Although there are a number of explanations (Costerton et al., 1999), the most powerful is the protection afforded by the slime matrix. This either physically limits penetration or, in the case of oxidising biocides such as sodium hypochlorite and peracetic acid, is deactivated in the biofilm's outer layers. Another argument builds on the likelihood that some parts of the biofilm are starved and, consequently, are in a more robust slow growing physiological state. There is also the view that some cells when present in a biofilm change their phenotype to become more resistant to antimicrobial agents. Whatever the mechanism, there is universal acceptance that micro-organisms are more robust in biofilms than when in the free, planktonic form. The degree of protection is by no means precise but has been estimated by numerous authors to be between 10 and 100-fold.

Unfortunately, in the real world there is a mindset that planktonic organisms are the enemy of hygiene rather than the 'sessile' micro-organisms in a biofilm. The real practical significance of this is brought out from work with biofilms in water distribution systems. Alarmingly, Keevil et al. (1995) reported that a free planktonic cell concentration of 10-103ml 1 is equivalent to an attached cell population of 105-107cm 2. This observation is of particular interest in the brewing industry where the hygienic status of closed vessels and mains is typically determined by measurement of the microbial loading in the residual rinse liquor. Although undeniably significant in validation of CiP operations, in many respects this insight is unfortunate! The perception that brewing microbiology is less than an exact science and is prone to ambiguity is further undermined by the microbiological loading of rinses effectively representing the tip of the microbial iceberg!

Explicit reports in the brewing press on the importance of biofilms to process and product hygiene have been limited to publications stemming from Diversey (Czechowski & Banner, 1992; Banner, 1994) and from VTT in Finland (Storgards et al., 1997b; 1999a-c). These groundbreaking studies have clearly shown that archetypal brewery contaminants are adept at forming biofilms on surface materials found in breweries. For example, Czechowski and Banner (1992) reported the ready attachment of E. agglomerans, an Acetobacter species (Table 8.1) and L. brevis (Table 8.2) to stainless steel and other materials. Similarly, Storgards et al. (1997a) demonstrated that 11 out of 20 bacterial species (Acetobacter species, Gluconono-bacter oxydans, L. lindneri, E. aggolerans) were capable of forming biofilms on stainless steel. All of the yeasts studied (a diastatic strain of S. cerevisiae, P. mem-branifaciens and B. anomalus) were found to attach to stainless steel and were classified as 'strong biofilm producers'.

The theme of attachment to some of the different surfaces found in the brewing process was investigated by Storgards et al. (1999a-c), some of whose images are presented in Fig. 8.11. Using a model closed circulating test rig, new and 'aged' coupons of polymeric materials found in gaskets and valves were compared to stainless steel in terms of supporting biofilm formation and 'cleanability' post CiP. With new materials (Storgards et al., 1999a) both E. agglomerans and P. inopinatus (Table 8.2) were found to attach readily to EPDM (ethylene propylene diene monomer rubber), PTFE (polytetrafluoroethylene) and Viton (fluoroelastomer). Presumably because of its bacteriostatic properties, new NBR (nitrile butyl rubber or

Fig. 8.11 Images of biofilms of yeast/bacteria (kindly provided by Erna Storgards. VTT. Helsinki. Finland).

Buna-N) was found to support little biofilm growth. In terms of hot CiP, the biofilms were more readily removed from NBR and PTFE than EPDM and Viton.

Outside of the brewery, the ability of micro-organisms to adhere to surfaces within draught beer dispense systems has long been recognised. In work that remains pertinent today, Harper (1981) described the presence of a variety of yeast genera in dispensed beer and noted the attachment of the same yeasts to the lumen surface of dispense pipes. Indeed, without naming them, the characteristics and threats of biofilms were succinctly described in a previous paper by Harper et al. (1980). Firstly, they noted that 'many of these organisms produce sticky substances enabling them to adhere readily'. The second point, which reinforces the threat and significance of biofilms, noted that 'if the sanitiser fails to penetrate their accumulations and fails to scour them from the walls, then cleaning is only going to be partially effective'.

The challenge of monitoring and removing attached organisms from surfaces is discussed elsewhere in this chapter.

8.1.4.2 Air and process gases. Air hygiene has attracted little attention in the brewing industry. This presumably reflects the view that it is of little importance in brewery hygiene. Either there is no risk as the process is contained within closed vessels or, in the case of open vessels, any threat is accepted as being minor. Although in principle correct, air is a potent source of micro-organisms, which in a brewery, have the capability to spoil or distort product quality. This is well demonstrated by the spoilage of unpasteurised cask beers in cellars (Section 8.1.4.1) and the production of Iambic and traditional French ciders by spontaneous fermentation.

The distinctive Iambic beers are produced in breweries around the town of Lem-beek in Belgium (see Section 2.2). Here hot wort is cooled overnight in shallow open trays whilst air is blown across the surface of the liquid. After transfer into wooden casks, mixed flora fermentation occurs over a two to three year period (van Oevelen et al., 1977; Martens et al., 1991). Although the bulk of the fermentation is performed by S. cerevisiae, K. apiculata (Table 8.3) and various Enterobacteriaceae (Table 8.1) (E. cloacae and K. aerogenes) predominate during the first month or so. After about eight months, the Saccharomyces are replaced by various non-Saccharomyces wild yeasts (Table 8.3). These include Brettanomyces species (B. bruxellensis and B. lambicus) and, to a lesser extent, other wild yeasts (Candida, Cryptococcus, Torulopsis and Pichia). In terms of bacteria, Pediococcus species (principally P. damnosus syn. cerevisiae) succeed the Enterobacteriaceae, and acetic acid bacteria, although less welcome, can be present throughout the latter part of the process. Similarly, the contribution of airborne contaminants is critical in the spontaneous fermentation of apple juice, in the production of traditional French ciders. Laplace et al. (1998) have clearly demonstrated the importance of the 'surrounding air' (and biofilms on 'utensils') in providing lactic acid bacteria for the malolactic fermentation in cider making.

The one area of brewing in which the threats of poor air hygiene have been recognised is that of sterile filtration and aseptic filling of beer. The growth of the non-pasteurised 'fresh' beers in Japan and the USA has required a step change in attitude and approach to product and environmental hygiene. Indeed Ryder et al. (1994) have described the need for a 'sterile envelope' to enclose the processing of sterile filtered beer and subsequent filling operations. The importance of air hygiene around the bottle or can filler is reinforced by the prescriptive standards of air conditioning in this area. Ryder et al. (1994) describe the use of high efficiency particulate air (HEPA) filters capable of removing 99.97% of all particles >0.2 (im to deliver air to these areas. In addition to air, the 'sanitary mindset' (Ryder et al., 1994) requires the hygienic management of all surfaces in the filler area together with infeeds such as conveyors, bottles and cans.

Although the threat of poor air hygiene in aseptic filling operations is a given, two publications from VTT in Finland (Henriksson & Haikara, 1991; Haikara & Hen-riksson, 1992) have sought to quantify the microbial risk. Using a portable 'SAS' device that can sample up to 180 litres of air in a minute, the microbial loading in the air was determined (in the autumn and spring) within the bottling halls often Finnish breweries. Analysis of anaerobic, mainly lactic acid bacteria in the air (see Table 8.4) identified the bottle filler as being a particular microbial hotspot. Of note was the observation that the airborne loading in breweries experiencing microbiological problems was three times that of other breweries without such concerns. Airborne yeasts were numerically lower (Table 8.4) and the loading was not influenced by season, unlike the anaerobes which were 2-6 times higher in the autumn than in the spring. As would be expected, this survey demonstrated that high airborne counts were associated with environments that typically had higher temperatures and humidity.

Table 8.4 Air hygiene in filling area (after Henriksson & Haikara, 1991).

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