isomers. Generally, Gram-positive bacteria are sensitive to these isomerised hop acids (see Table 8.2) and, accordingly cannot grow in hopped beers. However as noted in Section 8.1.1, strains of Lactobacillus (Fig. 8.7) and Pediococcus (Fig. 8.8) able to spoil beer are significantly more resistant to these acids. Work by Simpson and Fernandez (1992) showed great variation in the sensitivity of a selection of Gram negative bacteria to one of the major hop acids, /rans-isohumulone. The minimum inhibitory concentration (MIC) of /rans-isohumulone required to inhibit the growth of 42 Lactobacillus strains ranged from < 20 (iM (19% of strains) through 20-40 (iM (45%) to >80 |iM (24%). Those that could resist >80 |iM (120-180 |iM) trans-isohumulone 'had invariably been isolated from spoilt beer' (Simpson & Fernandez, 1992). Building on this, Simpson and Fernandez (1994) classified beer spoilage lactic acid bacteria as having a MIC of 90-200 (iM whereas sensitive, non-spoilage organisms had a MIC of 10-40 (iM. To put these results into perspective, the concentration of iso-a-acids in 'normal' UK commercial beers is in the range of 6070 (iM, which is equivalent to 20-25 mg/L of bitterness. However, as would be anticipated for weak acids, the MIC for iso-a-acids varies with pH. Simpson (1993) has reported that the antibacterial activity of /ra?w-isohumulone decreases 800-fold as

Fig. 8.7 Electron micrograph of Lactobacillus brevis (kindly provided by Bill Simpson of Cara Technology).
Fig. 8.8 Electron micrograph of Pediococcus danmosus (kindly provided by Bill Simpson of Cara Technology.

the pH is increased from 4 to 7. From a practical perspective a 'change in pH of as little as 0.2 can reduce the protective effect of hop compounds by as much as 50%' (Simpson, 1993).

Although many questions remain to be answered, the work of Simpson and colleagues has provided a solution to the practical issue of whether or not an isolate is a beer spoiler. Typically, lactic acid bacteria isolated from beer will not grow when the colony is transferred to beer. This is unsatisfactory, as the spoilage status of the isolate is not clear. Simpson and Fernandez (1992) resolved this issue by demonstrating that hop-resistant strains could be trained to grow in beer by pregrowth in media containing Zraras-isohumulone (45 (iM). Similarly, it is well known that spoilage lactic acid bacteria can be encouraged to eventually grow in beer by subculture into media containing an increasing ratio of beer. Conversely, hop-sensitive organisms pregrown in the presence of non-inhibitory concentrations (8 (iM) could not grow in beer. Although undeniably useful, such approaches are inevitably slow and cannot be applied in routine microbiological testing. A more practical application is in making microbiological media more selective for hop-resistant bacteria. Simpson and Hammond (1991) reported that the inclusion of 20 (iM Zraras-isohumulone in MRS media suppressed the growth of a sensitive Lactobacillus but enabled the growth of a resistant strain (Simpson & Hammond, 1991).

It is noteworthy that hop-sensitive and hop-resistant lactic acid bacteria are indistinguishable from each other in terms of morphology, physiology and metabolism (Fernandez & Simpson, 1993). A promising molecular explanation for hop resistance has been reported by Sami et al. (1997b). The presence or absence of a plasmid gene horA correlates strongly with, respectively, hop resistance or sensitivity. Of 61 strains, which were horA-positive strains, 59 could grow in beer. Conversely, only one of 34 horA-negative strains could grow in beer. Using PCR (see Section 4.2.6), beer spoilage capability (horA-PCR positive) can be assessed in about six hours.

Although not yet watertight, the observations of Sami et al. (1997b) may eventually explain the molecular basis of hop resistance in lactic acid bacteria. Certainly the observation (Sami et al., 1997a) that the horA gene found in hop-resistant strains of L. brevis has homology with multidrug resistance proteins in mammals and Lactococcus adds weight to the argument proposed by Sami et al. (1997b). Although speculative, the horA gene product may negate the impact of hop acids in resistant cells. A physiological spin on the metabolic impact of iso-a-acids on sensitive strains comes from the work of Simpson and his colleagues. In summary (see Fig. 8.9), isohumulones act as ionophores and catalyse the transport of ions across the bacterial membrane. In his Cambridge Prize review, Simpson (1993) has shown the addition of trans-isohumulone to a hop-sensitive strain to result in the import of protons into the cell with a concomitant reduction in intracellular pH. As maintenance of a proton gradient across the cell membrane is necessary for nutrient transport, such circumstances result in starvation and, ultimately, cell death.

8.1.3 Spoilage micro-organisms - yeast

A popular definition of 'wild' yeasts in the brewing industry is 'any yeast not deliberately used and under full control' (Gilliland, 1971a). The definition of wild

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