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In a subsequent report (Haikara & Henriksson, 1992), the level of anaerobes at the filler was noted as being indicative of the general air hygiene in the bottling hall. Not surprisingly, the filler air hygiene was reduced by improving the cleaning and disinfection regimes in this area. Further, the rich threat from the growth of microorganisms in beer-contaminated conveyor lubricant soaps was dramatically reduced by switching to synthetic lubricants containing biocides. Intriguingly, this study showed that the strictly anaerobic P. cerevisiiphilus (see Table 8.1) could be isolated from air around the filler.

Comparable but hitherto unpublished observations (Andrew Jones and David Quain) from a cask (unpasteurised) beer filling hall, suggests a similar airborne loading to those reported in Table 8.4. Here, the results (in July) ranged from 250 (cask washing) through 400 (filling) to 600 cfu m3 (conveyor leaving the hall). As with the Finnish observations, bacterial loading was greater than airborne yeasts. How ever, an airborne 'phenolic' wild yeast (P. membranifaciens - see Table 8.3) was clearly implicated in a previous 'trade problem' in cask beer from this brewery.

Inevitably, the sources of micro-organisms in the air are many and various! The loading of airborne micro-organisms can be assumed to reflect a series of equilibria between the availability of nutrients and appropriate micro-organisms able to exploit these nutrients together with distribution via movement of air. Therefore, not surprisingly, air hygiene reflects the general environment. Consequently, in a brewery, spillages of nutrient-rich soups, such as wort, beer and sugar syrups will act as nuclei for colonisation of settling airborne micro-organisms that can exploit such sources of nutrients. As they dry out these 'puddles of contamination' reseed the air with microorganisms, which go on to contaminate fresh spillages of nutrient-rich liquids.

Arguably, one of the most underestimated sources of airborne micro-organisms is the delivery of malt in the brewery. In busy breweries, this regular event punctuates the day with plumes of malt dust released into the air. As malt is laden with a variety of micro-organisms (Table 8.5), it would be anticipated that this dust is equally rich in bacteria and yeasts. Surely it is more than a coincidence that the micro-organisms recovered from barley and malt (O'Sullivan et al., 1999) are also typical microflora of beer. For example, the Gram-positive lactic acid bacteria found on malt include L. plantarum, L. fermentum, L. brevis and L. buchneri (Table 8.2). Similarly, the Gramnegative bacteria (Table 8.1) included Enterobacter agglomerans, Klebsiella pneumoniae, Rahnella aquitilis and Citrobacter freundii. Yeasts found on barley and malt (for a review see Flannigan, 1996) include such familiar genera as Rhodotorula, Hansenula, Candida and Torulopsis.

Table 8.5 Microbiology of kilned and screened malt (cfug malt ').

Makings

Aerobic bacteria

Lactic acid bacteria

Pseudomonads

Coliforms

Yeasts/ moulds

Source

Modern pneumatic (batch size 150 tonne)

1.7 X 107

1.7 xlO5

1.7 xlO5

7.5 xlO5

2.7 X 104

O'Sullivan et al. (1999)

Traditional floor (batch size 30 tonne)

5.0 X 105

8.0 xlO4

9.0 xlO3

2.0 xlO4

3.0 X 104

O'Sullivan et al. (1999)

Not reported

5.5 X 106

5.7 xlO4

1.9 X 104

Flannigan (1996)

From the perspective of product hygiene, the direct role of malt microflora is minimal. O'Sullivan et al. (1999) showed the loading of viable organisms to decline dramatically during mashing such that only very low numbers of lactic acid bacteria survive to the copper, where they are obviously killed! Whether malt microflora play a bigger role in brewery hygiene remains to be clarified. However, it is intriguing to speculate that every malt delivery effectively 'inoculates' the wider environment with micro-organisms that can survive alongside yeast, and spoil wort or beer. Although only a hypothesis, it is timely for a study, using molecular fingerprinting methods that can track strains of micro-organisms across the process, to resolve this fascinating concept. If malt microflora are implicated, as would seem likely, as being a threat to brewery hygiene, it will be necessary to identify routes to minimising the generation and distribution of malt dust during delivery and handling.

Although yet to be studied in brewery environment, the movement and distribution of air would be anticipated to play an important role in exacerbating or minimising the threat of poor air hygiene. Certainly, in the wider food industry, mapping airflows around process areas is increasingly recognised as a vehicle for understanding and minimising the microbial threat of airborne micro-organisms. It is noteworthy that airflow patterns can be reconfigured by demolition or the erection of buildings, installation and removal of vessels. In passing, such activities (particularly demolition) can be responsible for the generation and release of micro-organisms. Consequently, it is good practice to reduce any threat to air hygiene by partitioning and damping-down building works.

Although the significance of air hygiene is easily dismissed, particularly in breweries with closed vessels, it should be included in any consideration of 'total hygiene'. The threat is not limited to the high profile, more obvious threat of sterile filling operations but extends brewery-wide. Routes for the ingress and distribution of airborne micro-organisms into the general environment of the brewery should be understood and, where necessary controlled. Points of entry (such as manway doors) into 'closed' vessels should be identified, and managed to control the threat of entry.

Unlike air hygiene which, in the vast majority of breweries, does not feature in the microbiological sampling and testing plan, monitoring the microbiology of process gases is part of the normal QA plan (see Section 8.3.1.2). Process gases, such as nitrogen and carbon dioxide, have long been recognised as posing a threat through the colonisation of gas mains and subsequent transfer into product.

Although such gases are used across the process to minimise and control dissolved oxygen, the major threat is to bright, comparatively 'clean' beer prior to and including packaging. The most common concern is the practice of gas washing in bright beer tanks to reduce dissolved oxygen or to correct carbon dioxide and/or nitrogen concentration. Here, the duration of gas washing coupled with the vulnerability of bright beer make this process particularly susceptible to contamination by 'slugs' of contaminated gas. Similarly, microbiological contamination of process gases is a particular threat to product quality during packaging into bottles, cans and kegs.

Microbiological contamination of process gas is typically caused by beer finding its way into the gas lines. This can result in the colonisation of the gas main by anaerobic organisms, which by definition will be capable of beer spoilage. In terms of HACCP (see Section 8.2), management of process gases to remove or minimise the risk is particularly important. Indeed, process gas quality is a 'critical control point' where the risk is managed by regular cleaning and steam sterilisation. As a second line of defence, process gases are filtered at the point of use to prevent contamination. For this to be effective, the filters themselves must be periodically sterilised and subject to a maintenance schedule of inspection and replacement.

8.1.5 Food safety

In his Laurence Bishop Memorial Lecture, Long (1999) reviewed food safety issues that have challenged or are challenging the brewing industry. Two drivers are identified that have heightened consumer awareness of food safety. First, the consumer has been 'sensitised' through scares such as BSE in beef products, benzene in carbonated drinks, E. coli contamination of inadequately cooked foods and Salmonella in eggs. Second, analytical capability/detection is developing more rapidly than the associated understanding of the toxicological impact of the identified compounds. Indeed, Long (1999) notes that the focus should move to understanding the 'actual balance of toxic and protective components in the food matrix rather than to assess the toxicology of components in isolation'. Thankfully, from a microbiological perspective, there have been relatively few food safety concerns in the brewing industry. The most notable of these is the role of the yeast contaminant O. proteus in the formation of non-volatile nitrosamines in beer.

8.1.5.1 Nitrosamines. In the late 1970s, N-nitrosamines were found in cured meat and malt beverages (for a review see Smith, 1994). This was of great concern as nitrosamines are powerful carcinogens in animal systems and there is 'compelling' but indirect evidence of involvement in human cancers (Long, 1999). Consequently, the brewing industry rapidly sought to understand the mechanism of formation of nitrosamines and to eliminate the risk. Control of the volatile N-nitrosamines was achieved through changes in the malt kilning process, whereas the non-volatile nitrosamines (apparent total N-nitroso compounds - ATNC) necessitated improvements in pitching yeast hygiene.

Work in the late 1980s primarily at the Brewing Research Foundation (now Brewing Research International) unravelled the pathway of ATNC formation during brewery fermentation. In short (Fig. 8.12), nitrate is reduced by O.proteus to nitrite which reacts with wort amines to produce ATNC. As nitrate (from liquor or hops) is inevitably present in worts, the only control strategy is to eliminate O. proteus from the equation through improved brewery hygiene and/or acid washing (see Sections 7.3.3 and 8.2.2).

O. proteus is a Gram-negative member of the Enterobacteriaceae (see Table 8.1)

liquor, hops

Fig. 8.12 Formation of ATNCs during fermentation.

which, according to Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) is closely related to the Escherichia genus. Although found in cooled wort, it is often described as the 'short fat rod of pitching yeast' (van Vuuren, 1996) with which it co-sediments and is recycled. Growth of O. proteus is encouraged by the reduction of nitrate to nitrite, which inhibits yeast growth and slows fermentation. Numerically, contamination of pitching yeast with greater than 0.03% O. proteus results in ATNC exceeding the Brewers' Society (now Brewers and Licensed Retailers Association -BLRA) recommended limit of 20 (igl 1 (Calderbank & Hammond, 1989).

The quantitative interrelationships between nitrate, nitrite and ATNC were demonstrated by Calderbank and Hammond (1989). Figure 8.13 graphically shows the reduction of nitrate, appearance of nitrite and ATNC during fermentation. Conversion of nitrite to ATNC is by no means quantitative, such that for every lOmgl 1 nitrate reduced about 90 j-ig 1 1 ATNC is formed. This is believed to reflect the loss of nitrite (as nitrous acid) through gas stripping (Simpson et al., 1988) and pH dependency of N-nitrosation of amines (Smith, 1994).

Fig. 8.13 Formation of nitrite (♦) and ATNC (A) in nitrate (■) supplemented laboratory fermentations contaminated with O. proteus. Concentration of nitrate/nitrite in mgL1 and ATNC in jjgl 1. Data from Calderbank and Hammond (1989).

Fig. 8.13 Formation of nitrite (♦) and ATNC (A) in nitrate (■) supplemented laboratory fermentations contaminated with O. proteus. Concentration of nitrate/nitrite in mgL1 and ATNC in jjgl 1. Data from Calderbank and Hammond (1989).

An alternative, less well-recognised route to the formation of ATNC involves Gram-positive Bacillus species (Smith, 1994). As they are sensitive to hop acids, these contaminants are typically found in the brewhouse in sweet wort and hot brewing liquor. One, B. coagulans, has been shown to produce ATNCs in hot wort (30-70°C) at a rate which is ten times faster than O. proteus (Smith et al., 1992). These organisms are notable for their thermotolerance and 'slimy', glutinous appearance which makes them excellent candidates for involvement in surface biofilms (see Section 8.1.4.1).

Smith also investigated the possibility that brewery wild yeasts might provide a further route for ATNC formation (Smith, 1992). Although capable of nitrate reduction in model experiments, ammonia and amino acids were shown to repress this reaction. In brewery practice, contaminating yeasts such as Hansenula anómala and Rhodotorula glutinis are unable to reduce nitrate and consequently are unlikely to provide a further microbial route to ATNC in beer.

8.1.5.2 Biogenic amines. Elevated levels of biogenic amines in cheese, meat and fish products have been linked with a variety of undesirable physiological reactions. Specifically, high levels of histamine are associated with 'histaminic intoxication' and tyramine has been linked with food induced migraines. The major biogenic amines found in beers and their typical concentrations are reported in Table 8.6. Although considered 'too low to produce direct toxicological effects' (Izquierdo-Pulido et al., 1996b), there have been sporadic reports of toxic reactions to beer consumption which have implicated biogenic amines, specifically tyramine. In particular, severe hypertensive events after the consumption of 'modest' amounts of draught beer have been described for two individuals in Canada being treated with monoamine oxidase inhibitors (MAOI) (Tailor et al., 1993; Shulman et al., 1997). Treatment with MAOIs is important in the management of a variety of clinical conditions including depression. However, as hypertensive crises can be triggered by MAOIs and biogenic amines, patients require a specially reduced tyramine diet to minimise the threat of MAOI mediated elevation in blood pressure.

Table 8.6 Biogenic amines in beer. Data for 195 European canned or bottle beers from Izquierdo-Pulido et al. (1996b).

Biogenic amine

Range (mgl ')

Mean (mg 1 ')

Histamine

Not detected (nd)-21.6

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