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Review article
a
Fundamental Research Laboratory, Asahi Breweries, Ltd. 1-21, Midori 1-chome, Moriya-shi, Ibaraki 302-0106, Japan
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30,
9751NN Haren, The Netherlands
Abstract
For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such
as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as
Pectinatus cerevisiiphilus, Pectinatus frisingensis and Megasphaera cerevisiae. They can spoil beer by turbidity, acidity and the
production of unfavorable smell such as diacetyl or hydrogen sulfide. For the microbiological control, many advanced
biotechnological techniques such as immunoassay and polymerase chain reaction (PCR) have been applied in place of the
conventional and time-consuming method of incubation on culture media. Subsequently, a method is needed to determine
whether the detected bacterium is capable of growing in beer or not. In lactic acid bacteria, hop resistance is crucial for their
ability to grow in beer. Hop compounds, mainly iso-a-acids in beer, have antibacterial activity against Gram-positive bacteria.
They act as ionophores which dissipate the pH gradient across the cytoplasmic membrane and reduce the proton motive force
(pmf). Consequently, the pmf-dependent nutrient uptake is hampered, resulting in cell death. The hop-resistance mechanisms in
lactic acid bacteria have been investigated. HorA was found to excrete hop compounds in an ATP-dependent manner from the
cell membrane to outer medium. Additionally, increased proton pumping by the membrane bound H+-ATPase contributes to
hop resistance. To energize such ATP-dependent transporters hop-resistant cells contain larger ATP pools than hop-sensitive
cells. Furthermore, a pmf-dependent hop transporter was recently presented. Understanding the hop-resistance mechanisms has
enabled the development of rapid methods to discriminate beer spoilage strains from nonspoilers. The horA-PCR method has
been applied for bacterial control in breweries. Also, a discrimination method was developed based on ATP pool measurement
in lactobacillus cells. However, some potential hop-resistant strains cannot grow in beer unless they have first been exposed to
subinhibitory concentration of hop compounds. The beer spoilage ability of Pectinatus spp. and M. cerevisiae has been poorly
studied. Since all the strains have been reported to be capable of beer spoiling, species identification is sufficient for the
breweries. However, with the current trend of beer flavor (lower alcohol and bitterness), there is the potential risk that not yet
reported bacteria will contribute to beer spoilage. Investigation of the beer spoilage ability of especially Gram-negative bacteria
may be useful to reduce this risk.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Beer; Spoilage; Bacteria; Detection; Hop; Antibacterial; Resistance
* Corresponding author. Fundamental Research Laboratory, Asahi Breweries, Ltd. 1-21, Midori 1-chome, Moriya-shi, Ibaraki 302-0106,
Japan. Tel.: +81-297-46-1504; fax: +81-297-46-1506.
E-mail address: kanta.sakamoto@asahibeer.co.jp (K. Sakamoto).
0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0168-1605(03)00153-3
106
1. Introduction
Beer has been recognized for hundreds of years as
a safe beverage. It is hard to spoil and has a remarkable microbiological stability. The reason is that beer
is an unfavorable medium for many microorganisms
due to the presence of ethanol (0.5 10% w/w), hop
bitter compounds (approximately 17 55 ppm of isoa-acids), the high content of carbondioxide (approximately 0.5% w/w), the low pH (3.8 4.7), the extremely reduced content of oxygen ( < 0.1 ppm) and
the presence of only traces of nutritive substances
such as glucose, maltose and maltotriose. These latter
carbon sources have been substrates for brewing yeast
during fermentation. As a result, pathogens such as
Salmonellae typhimurium and Staphylococcus aureus
do not grow or survive in beer (Bunker, 1955).
However, in spite of these unfavorable features, a
few microorganisms still manage to grow in beer.
These, so-called beer spoilage microorganisms, can
cause an increase of turbidity and unpleasant sensory
changes of beer. Needless to say that these changes
can affect negatively not only the quality of final
product but also the financial gain of the brewing
companies.
A number of microorganisms have been reported
to be beer spoilage microorganisms, among which
both Gram-positive and Gram-negative bacteria, as
well as so-called wild yeasts. Gram-positive beer
spoilage bacteria include lactic acid bacteria belonging to the genera Lactobacillus and Pediococcus.
They are recognized as the most hazardous bacteria
for breweries since these organisms are responsible
for approximately 70% of the microbial beer-spoilage
incidents (Back, 1994). The second group of beer
spoilage bacteria is Gram-negative bacteria of the
genera Pectinatus and Megasphaera. The roles of
these strictly anaerobic bacteria in beer spoilage have
increased since the improved technology in modern
breweries has resulted in significant reduction of
oxygen content in the final products. Wild yeasts do
cause less serious spoilage problem than bacteria but
are considered a serious nuisance to brewers because
of the difficulty to discriminate them from brewing
yeasts.
Considerable effort has been made by many microbiologist to control microbial contamination in
beer. The most commonly used method today for
Table 1
Beer spoilage bacteria
Gram-positive
bacteria
107
Gram-negative
bacteria
Rod-shaped
Cocci
Lactobacillus spp.
Lb. brevis
Lb. brevisimilis
Lb. buchneri
Lb. casei
Lb. coryneformis
Lb. curvatus
Lb. lindneri
Lb. malefermentans
Lb. parabuchneri
Lb. plantarum
Pectinatus spp.
P. cerevisiiphilus
P. frisingensis
P. sp. DSM20764
Selenomonas sp.
S. lacticifex
Zymophilus sp.
Z. raffinosivorans
Pediococcus spp.
P. damnosus
P. dextrinicus
P. inopinatus
Micrococcus sp.
M. kristinae
Megasphaera sp.
M. cerevisiae
Zymomonas sp.
Z. mobilis
108
appears to be the most important beer spoiling Lactobacillus species and is detected at high frequency in
beer and breweries. More than half of the bacterial
incidents were caused by this species (Back et al.,
1988; Back, 1994; Hollerova and Kubizniakova,
2001). Lb. brevis is an obligate heterofermentative
bacterium. It is one of the best-studied beer spoilage
bacteria and grows optimally at 30 jC and pH 4 6
and is generally resistant to hop compounds. It is
physiologically versatile and can cause various problems in beer such as super-attenuation, due to the
ability to ferment dextrins and starch (Lawrence,
1988). The antibacterial effects of hop compounds
and the mechanism(s) responsible for hop resistance
have been studied in detail in this species (Simpson,
1991, 1993a,b; Simpson and Smith, 1992; Fernandez
and Simpson, 1993; Simpson and Fernandez, 1994;
Sami et al., 1997a,b, 1998; Sakamoto et al., 2001,
2002; Suzuki et al., 2002).
The second most important beer spoiling lactobacillus, Lactobacillus lindneri is responsible for 15
25% of beer-spoilage incidents (Back et al., 1988;
Back, 1994). The physiology of Lb. lindneri is very
similar to that of Lb. brevis and only recently has Lb.
lindneri been recognized on the basis of its 16S rRNA
gene sequence as a phylogenetically separate species
in the genus Lactobacillus (Back et al., 1996; Anon.,
1997). Lb. lindneri is highly resistant to hop compounds (Back, 1981) and grows optimally at 19 23
jC (Priest, 1987; Back et al., 1996) but survives
higher thermal treatments than other lactic acid bacteria (Back et al., 1992). All Lb. lindneri strains, tested
so far, are capable of beer spoilage, while other
lactobacilli comprise both beer spoiling and nonspoiling strains (Rinck and Warkerbauer, 1987a,b; Storgards et al., 1998).
Lactobacillus buchneri, Lb. casei, Lb. coryneformis, Lb. curvatus and Lb. plantarum are less common
beer-spoiling bacteria than the two species described
above (Priest, 1996). Lb. buchneri resembles closely
Lb. brevis but differs in its ability to ferment melezitose. In addition, some Lb. buchneri strains require
riboflavin in their growth medium (Sharpe, 1979;
Back, 1981). Lb. casei can produce diacetyl, which
gives beer an unacceptable buttery flavor. Diacetyl
appears to be more potent in that respect than lactic
acid, the major end-product of lactic acid bacteria.
The threshold value of diacetyl in beer is much lower
109
2.2.1. Pectinatus
Pectinatus spp. are now recognized as one of the
most detestable beer spoilage bacteria. They play a
major role in 20 30% of bacterial incidents, mainly in
nonpasteurized beer rather than in pasteurized beer
(Back, 1994). Pectinatus species were long thought to
be Zymomonas spp. because of their phenotypical
similarities. The first isolate was obtained from breweries in 1971 (Lee et al., 1978) and so were all
subsequent isolates (Back et al., 1979; Haukeli,
1980; Kirchner et al., 1980; Haikara et al., 1981;
Takahashi, 1983, Soberka et al., 1988, 1989). The
natural habitat of the Pectinatus species are still
unknown (Haikara, 1991). Two species are found in
this genus: Pectinatus cerevisiiphilus and Pectinatus
frisingensis (Schleifer et al., 1990). There is also one
2.2.2. Megasphaera
Megasphaera has emerged in breweries along with
Pectinatus and is responsible for 3 7% of bacterial
beer incidents (Back et al., 1988; Back, 1994). They
are nonspore-forming, nonmotile, mesophilic cocci
that occur singly or in pairs and occasionally as short
chains. This genus includes two species, M. elsdeni
and M. cerevisiae. Since the first isolations in 1976,
only M. cerevisiae has been blamed to be responsible
for beer spoilage (Weiss et al., 1979; Haikara and
Lounatmaa, 1987; Lee, 1994). M. cerevisiae grows
between 15 and 37 jC with an optimum at 28 jC and
at pH values above 4.1. The growth is inhibited at
ethanol concentrations above 2.8 (w/v) but is still
possible up to 5.5 (w/v) (Haikara and Lounatmaa,
1987; Lawrence, 1988). It is the most anaerobic
110
fermentation process. Beer produced with yeasts contaminated with H. protea has a parsnip-like or fruity
odor and flavor (van Vuuren, 1996). Abnormally high
levels of diacetyl and dimethly sulfide were detected
in beer produced from wort contaminated by R.
aquatilis (van Vuuren, 1996).
Recently, a novel strictly anaerobic Gram-negative
bacterium was isolated from a brewery (Nakakita et
al., 1998). It is a rod-shape bacterium with no flagella
that can grow in beer at pH values above 4.3 and does
not produce propionic acid. Genetic and phenotipical
studies indicated that this bacterium is different from
Pectinatus, Zymomonas and Selenomonas spp.
Recommended bya
Bacteria
b
LAB
LAB, G(
LAB
LAB
EBC, BCOJ
LAB
EBC, BCOJ
LAB
LAB
EBC, BCOJ
EBC, BCOJ
G(
G(
)
)
EBC, BCOJ
EBC, BCOJ
G( )
G( )
LAB, G(
LAB, G(
)
)
EBC
EBC, BCOJ
EBC, ASBC, BCOJ
EBC, BCOJ
LAB, G(
ASBC
LAB
ASBC
LAB
ASBC
G(
ASBC, BCOJ
111
112
113
114
Fig. 2. Chemical structures of hop compounds. The name of each a-acid (I) and h-acid (II) is dependent on its side chain. During wort boiling
process, a-acids (naturally R-body; III) are isomerized to result in stereoisomers of trans-isohumulones (IV) and cis-isohumulones (V).
115
116
resistance to ( )-humulone and colupulone (Fernandez and Simpson, 1993), suggesting a common mechanism of resistance against a broad range of hop acids.
trans-Isohumulone and ( )-humulone have three
hydrophobic side chains while colupulone has four.
The side chains are attached to a five-membered ring
of trans-isohumulone, but to a six-membered ring of
( )-humulone and colupulone (Fig. 2). On the other
hand, resistance was not observed to other ionophores
(nigericin, A23187, CCCP, monesin), weak acid food
preservatives (sorbic acid, benzoic acid), solvents
(ethanol) or antibiotics (ampicillin, cefamandole, vancomycin) (Fernandez and Simpson, 1993). The resistance mechanism might be specific for the h-triketone
group of the hop acids, which plays an essential role
in the antibacterial action (Simpson, 1991).
Microorganisms have developed various ways to
resist the toxicity of antibacterial agents:
(i) enzymatic drug inactivation. A well known
example is beta-lactamase which hydrolyzes the beta-lactam ring into innocuous substrates. In hop-resistant strains of Lb. brevis, neither conversion nor
inactivation of trans-isohumulone was found (Simpson and Fernandez, 1994).
(ii) target alteration. Cellular targets can be altered
by mutation or enzymatic modification in such a way
that the affinity of the target for the antibiotics is
reduced. In the case of trans-isohumulone, the target
site is the cell membrane (Teuber and Schmalreck,
1973; Schmalreck et al., 1975; Simpson, 1993a,b). A
sake (Japanese rice wine) spoilage bacterium, Lb.
heterohiochii, contains extremely long-chain fatty
acids in its membrane (Uchida, 1974), which may
play a role in ethanol resistance (Ingram and Buttke,
1984). It is possible but not yet investigated that hopresistant Lb. brevis has also a changed lipid composition of its membrane to lower the permeability to
hop compounds.
(iii) inhibition of drug influx. The outer membrane
of Gram-negative bacteria restricts the permeation of
lipophilic drugs, while the cell wall of the Grampositive mycobacteria has been found to be an exceptionally efficient barrier. The permeation of hop compounds might also be affected by the presence of a
galactosylated glycerol teichoic acid in beer spoilage
lactic acid bacteria (Yasui and Yoda, 1997b).
(iv) active extrusion of drugs. The presence of
(multi) drug resistance pump in the cytoplasmic
117
Fig. 4. Mechanisms of hop resistance. Hop compounds act as ionophores that exchange protons for cellular divalent cations. In a hop-sensitive
cell, hop compounds (Hop-H) invade the cell and dissociate into hop anions and protons due to the higher internal pH. Hop anions trap divalent
cations such as Mn2 + and diffuse out of the cell. The ionophoric action together with the diffusion of the hop metal complex results in an
electroneutral exchange of cations. Release of protons from hop compounds decreases the intracellular pH and results in a dissipation of the
transmembrane proton gradient (DpH) and the proton motive force (pmf). Consequently, pmf-driven uptake of nutrients will be decreased. In
hop-resistant cells, hop compounds can be expelled from the cytoplasmic membrane by HorA (a) (Sakamoto et al., 2001) and probably also by a
pmf-dependent transporter (b) (Suzuki et al., 2002). Furthermore, overexpressed H+-ATPase increases the pumping of protons released from the
hop compounds (c) (Sakamoto et al., 2002). More ATP is generated in hop-resistant cells than in hop-sensitive cells (Simpson and Fernandez,
1994). Galactosylated glycerol teichoic acid in the cell wall (Yasui et al., 1997) and a changed lipid composition of the cytoplasmic membrane
of beer spoilage lactic acid bacteria may increase the barrier to hop compounds.
118
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