International Journal of Food Microbiology 89 (2003) 105 – 124

www.elsevier.com/locate/ijfoodmicro

Review article
Beer spoilage bacteria and hop resistance
Kanta Sakamoto a,b,*, Wil N. Konings b
a
Fundamental Research Laboratory, Asahi Breweries, Ltd. 1-21, Midori 1-chome, Moriya-shi, Ibaraki 302-0106, Japan
b
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30,
9751NN Haren, The Netherlands
Received 4 September 2002; accepted 15 March 2003

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 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
1. Introduction
Beer has been recognized for hundreds of years as
a safe beverage. It is hard to spoil and has a remark-
able 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 iso-
a-acids), the high content of carbondioxide (approx-
imately 0.5% w/w), the low pH (3.8 –4.7), the ex-
tremely 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 belong-
ing 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 mi-
crobiologist to control microbial contamination in
beer. The most commonly used method today for
detecting beer spoilage bacteria in breweries is still
traditional incubation on culture media. A number of
selective media have been developed since Louis
Pasteur published in 1876 ‘Études sur La Biére
(Studies on beer)’ (Pasteur, 1876). It usually takes a
week or even longer for bacteria to form visible
colonies on plates or to increase the turbidity in
nutrients broths. Consequently, the products are often
already released for sale before the microbiological
results become available. If a beer spoilage bacterium
is then detected and identified in the beer product it
needs to be recalled from the market. This will cause
serious commercial damages to the brewery. Most
microbiologists have focused on developing more
specific and rapid methods for the detection of beer
spoilage bacteria than using traditional culture meth-
ods. However, a method is needed to determine
whether the bacteria detected are capable of growing
in beer or not. Up to date, the only available method is
the so-called ‘forcing test’ in which the detected
bacterium is re-inoculated and incubated in beer. It
usually takes 1 month or even longer to detect visible
turbidity in the inoculated beer, meaning that this
method is not very practical. A rapid method to
predict beer-spoiling ability is therefore urgently
needed and the development of such a method will
be essential for understanding the nature of the beer-
spoiling ability.
Among the components of beer, hop compounds
have received a lot of attention for reason of their
preservative values and their bitterness. For centuries
it was generally believed that hops protect beer from
infection by most organisms, including pathogens, but
it was only in the 20th century that Shimwell
(1937a,b) showed that hop compounds only inhibit
growth of Gram-positive bacteria and not of Gram-
negative bacteria. His findings had a great impact
because many pathogens such as Salmonella species
are Gram-negative bacteria. Feature(s) such as low pH
and alcohol content undoubtedly have a negative
effect on growth of these pathogens in beer. Among
Gram-positive bacteria, some species of lactic acid
bacteria are less sensitive to hop compounds and are
able to grow in beer. Insight in the mechanism of
resistance of lactic acid bacteria to hop compounds is
important for understanding their beer-spoiling ability.
The antibacterial activity of hop compounds and the
hop resistance of lactic acid bacteria have been
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124 107

investigated. On the other hand, little studies have Table 1

been done on the beer-spoiling ability of Gram- Beer spoilage bacteria
negative bacteria. Rod-shaped Cocci
This report reviews the currently available infor- Gram-positive Lactobacillus spp. Pediococcus spp.
mation about beer spoilage bacteria, their growth and bacteria Lb. brevis P. damnosus
Lb. brevisimilis P. dextrinicus
spoilage activity in beer.
Lb. buchneri P. inopinatus
Lb. casei
Lb. coryneformis Micrococcus sp.
2. Beer spoilage bacteria Lb. curvatus M. kristinae
Lb. lindneri
Lb. malefermentans
Beer is a poor and rather hostile environment for
Lb. parabuchneri
most microorganisms. Its ethanol concentration ranges Lb. plantarum
from 0.5% to 10% (w/w) and is usually around 4– 5%. Gram-negative Pectinatus spp. Megasphaera sp.
Beer is usually acid with pH’s ranging from pH 3.8 to bacteria P. cerevisiiphilus M. cerevisiae
4.7, which is lower than most bacteria can tolerate for P. frisingensis
P. sp. DSM20764
growth. Furthermore, the high carbondioxide concen-
Selenomonas sp. Zymomonas sp.
tration (approximately 0.5% w/w) and extremely low S. lacticifex Z. mobilis
oxygen content ( < 0.1 ppm) makes beer a near to Zymophilus sp.
anaerobic medium. Z. raffinosivorans
Beer also contains bitter hop compounds (approx-
imately 17 – 55 ppm of iso-a-acids), which are toxic,
especially for Gram-positive bacteria. The concen- of species and genera of Gram-positive bacteria. Only
trations of nutritive substances, such as carbohy- a few of these lactic acid bacteria are beer-spoiling
drates and amino acids, are very low since most organisms. Most hazardous for the brewing industry
have been consumed by brewing yeasts during are those belonging to the genera Lactobacillus and
fermentation. Pediococcus. In the period 1980– 1990, 58– 88% of
Only a few bacteria are able to grow under such the microbial beer-spoilage incidents in Germany
inhospitable conditions and are able to spoil beer (see were caused by lactobacilli and pediococci (Back et
Table 1). These bacteria include both Gram-positive al., 1988; Back, 1994). Also in Czech all beer spoilage
and Gram-negative species. Gram-positive beer spoil- bacteria detected in the breweries belonged to lactic
age bacteria belong almost always to the lactic acid acid bacteria (Hollerová and Kubizniaková, 2001).
bacteria. They are regarded as most harmful for The situation in other countries seems to be similar
brewing industry and are the cause of most of bacte- although for commercial reasons little statistical in-
rial spoilage incidents. Only a few Gram-negative formation has been supplied. These lactic acid bacte-
bacteria are known to cause beer spoilage. Some of ria spoil beer by producing haze or rope and cause
these belong to the acetic acid bacteria and had unpleasant flavor changes such as sourness and atyp-
received most attention. Today, these aerobic bacteria ical odor.
do not present a serious problem in beer spoilage
anymore since improved brewing technology has led 2.1.1. Lactobacilli
to a drastic reduction of the oxygen content in beer. The genus Lactobacillus is the largest genus of
Instead, strictly anaerobic bacteria, typically Pectina- lactic acid bacteria and includes numerous species.
tus spp. and Megasphaera cerevisiae, have become They are widely used in various fermentation pro-
serious beer spoilage bacteria. cesses, including food products such as beer, wine,
yoghurt and pickles. In contrast to the general believe
2.1. Gram-positive bacteria that all lactobacilli can grow in beer, only a few
species have been reported to be capable of beer
Almost all the beer spoilage Gram-positive bacteria spoilage (Rainbow, 1981; Priest, 1987, 1996; Jes-
belong to lactic acid bacteria. They are a large group persen and Jakobsen, 1996). Lactobacillus brevis
108 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
appears to be the most important beer spoiling Lac-
tobacillus 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; Hollerová and Kubizniaková,
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 prob-
lems 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 lactoba-
cillus, 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 com-
pounds (Back, 1981) and grows optimally at 19 –23
jC (Priest, 1987; Back et al., 1996) but survives
higher thermal treatments than other lactic acid bac-
teria (Back et al., 1992). All Lb. lindneri strains, tested
so far, are capable of beer spoilage, while other
lactobacilli comprise both beer spoiling and nonspoil-
ing strains (Rinck and Warkerbauer, 1987a,b; Stor-
gårds et al., 1998).
Lactobacillus buchneri, Lb. casei, Lb. corynefor-
mis, 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 melezi-
tose. 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
(0.15 ppm) than of lactic acid (300 ppm) (Hough et
al., 1982). Diacetyl is also produced during normal
fermentation by yeast and too high levels of diacetyl
are produced when yeast is not properly removed
from young beer (Inoue, 1981). The pathway of
diacetyl formation by lactic acid bacteria has been
studied in detail (Speckman and Collins, 1968, 1973;
Jönsson and Pettersson, 1977).
Lactobacillus brevisimilis (Back, 1987), Lb. mal-
efermentans, Lb. parabuchneri (Farrow et al., 1988),
Lb. collinoides and Lb. paracasei subsp. paracasei
(Hallerová and Kubizniaková, 2001) have also been
reported to be beer spoilage species.
Recently, few additional newly discovered species
of lactobacilli have been added to the list of beer
spoilers. According to its 16S rRNA gene sequence, a
strain called BS-1 is related to Lb. coryneformis, but
its narrow fermentation pattern (only limited to glu-
cose, mannose and fructose) differs significantly not
only from that of Lb. coryneformis but also from other
Lactobacillus spp. (Nakakita et al., 1998). Two other
novel lactobacillus species were found in spoiled beer
with significantly different taxonomical properties
from those of other Lactobacillus spp. (Funahashi et
al., 1998). One species, LA-2, with a 16S rRNA gene
sequence 99.5% similar to that of Lb. collinoides, has
a strong beer soiling ability. The other species, LA-6,
has a weak beer-spoiling ability an did not show any
significant homology to the other Lactobacillus spp.
(Funahashi et al., 1998).
2.1.2. Pediococci
Pediococci are homofermentative bacteria which
grow in pairs and tetrads. They were originally known
as ‘sarcinae’ because their cell organization was
confused with that of true sarcinae. Beer spoilage,
caused by cocci and characterized by acid formation
and buttery aroma of diacetyl, was therefore called
‘sarcinae sickness’. Pediococcus spp. produce rope
and extensive amounts of diacetyl like Lb. casei. They
are found at many stages in the brewing process from
wort till finished beer. Several Pediococcus spp. have
been found in breweries: Pediococcus acidilactici,
Pediococcus damnosus P. dextrinicus, P. halophilus
which recently is classified as Tetragenococcus hal-
ophilus (Collins et al., 1990), P. inopinatus, P. parvu-
lus and P. pentosaceus (Back, 1978; Back and
Stackebrandt, 1978). Among them is P. damnosus,
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
the most common beer spoiler. It was responsible for
more than 20% of all bacterial incidents in the period
1980 –1987 (Back, 1980; Back et al., 1988) but only
for 3 – 4% in 1992 –1993 (Back, 1994). The incidence
of beer spoilage by pediococci has decreased most
likely as a result of improved sanitation conditions in
breweries. P. damnosus is generally resistant to hop
compounds. It is interesting that P. damnosus is
commonly found in beer and late fermentations, but
seldom in pitching yeast. In contrast, P. inopinatus is
frequently detected in pitching yeast but rarely in the
other stages of beer fermentations (McCaig and
Weaver, 1983; Priest, 1987). P. inopinatus and P.
dextrinicus can grow in beer at pH values above 4.2
and with low concentrations of ethanol and hop
compounds (Lawrence, 1988). P. pentsaceus and P.
acidilactici have never been reported to cause any
defect in finished beer (Simpson and Taguchi, 1995).
2.1.3. Other Gram-positive bacteria
In additional to Lactobacillus and Pediococcus
species, also, a species from the genus Microcococcus
has been reported to be occasionally responsible for
beer spoilage. M. kristinae can grow in beer with low
ethanol and hop compounds at pH values above 4.5
(Back, 1981). Micrococci are usually strictly aerobic
but M. kristinae can grow also under anaerobic
condition (Lawrence and Priest, 1981). It produces a
fruity atypical aroma in beer (Back, 1981).
2.2. Gram-negative bacteria
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 brew-
eries 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
109
stain, DSM20764, isolated from spoiled beer that
differs considerably in genotype from the two other
species (Weiss, personal communication, 1987). Its
16S rRNA gene sequence is distinctly different from
that of the other two species (Sakamoto, 1997).
Pectinatus spp. are nonspore-forming motile rods with
lateral flagella attached to the concave side of the cell
body. They swim actively and appear X-shaped when
cells are young and snake-like and longer when cells
are older. They possess some features that are char-
acteristic for Gram-positive bacteria (Haikara et al.,
1981) and are regarded as being intermediate between
Gram-positive and Gram-negative bacteria. Growth
takes place between 15 and 40 jC with an optimum
around 32 jC (Chelak and Ingledew, 1987), between
pH 3.5 and 6 with an optimum at 4.5 and in media
containing up to 4.5% (w/w) of ethanol (Chelak and
Ingledew, 1987; Watier et al., 1993). During growth,
considerable amounts of propionic and acetic acids
are produced as well as succinic and lactic acids and
acetoin. Pectinatus spp. can also ferment lactic acid.
Since lactic acid is the sole source of carbon in SMMP
medium (see Section 3.1), this medium is used for the
selective isolation of Pectinatus spp. and Megaspaera
spp. (Lee, 1994). The most characteristic feature of
spoilage caused by Pectinatus spp. is extensive tur-
bidity and an offensive ‘rotten egg’ smell brought by
the combination of various fatty acids, hydrogen
sulfide and methyl mercaptan (Lee et al., 1978,
1980; Haikara et al., 1981). This spoilage activity
can cause serious damages for breweries.
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 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
species known to exist in the brewing environment
(Seidel et al., 1979). Beer spoilage caused by this
organism results in a similar extreme turbidity as
Pectinatus and the production of considerable quan-
tities of butyric acid together with smaller amounts of
acetic, isovaleric, valeric and caproic acids as well as
acetoin (Seidel et al., 1979). Like Pectinatus, the
production of hydrogen sulfide causes a fecal odor
in beer (Lee, 1994), which makes this bacterium one
of the most feared organisms for brewers.
2.2.3. Other Gram-negative bacteria
In addition to the two genera described above,
some other Gram-negative bacteria have been found
to cause problems in the brewing industry. Anaerobic
Zymomonas spp. have been found in primed beer to
which sugar was added and in ale beer. Zymomonas
mobilis is an aerotolerant anaerobe and grows above
pH 3.4 and at ethanol concentrations below 10% (w/v)
(van Vuuren, 1996). There is no report of Z. mobilis
spoilage in lager beer, probably because of its selec-
tive fermentation character (nonfermentative of malt-
ose, maltotriose but fermentative of glucose, fructose
and sucrose). It produces high levels of acetaldehydes
and hydrogen sulfide. Also another Zymophilus spp.,
Z. raffinosivorans, has been reported as a beer spoiler
(Schleifer et al., 1990; Seidel-Rüfer, 1990). The genus
Zymophilus is phylogenetically close to the genus
Pectinatus. Zymophilus spp. can grow in beer, like
Pectinatus spp., at pH values above 4.3 – 4.6 and
ethanol concentrations below 5% (w/v). Also, their
beer spoilage activity is similar to that of Pectinatus
spp. (Jespersen and Jakobsen, 1996).
Another Gram-negative bacterium Selenomonas
lacitifex has also been reported to play a role in
certain beer spoilage incidents but this species has
hardly been studied (Schleifer et al., 1990). Histori-
cally, a lot of attention of the brewing industry has
been given to aerobic Gram-negative bacteria. Acetic
acid bacteria, i.e., Gluconobacter and Acetobacter
used to be well known to breweries. They convert
ethanol into acetate, which results in vinegary off-
flavor of beer. For reasons explained above, such
aerobes are no longer important in modern breweries.
Hafnia protea, formerly Obesumbacterium prote-
us, and Rahnella aquatilis, formerly Enterobacter
agglomerans, have been detected in pitching yeasts
but never in finished beer. They can retard the
fermentation process. Beer produced with yeasts con-
taminated 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.
3. Detection of beer spoilage bacteria
3.1. Culture media
It has been seen above that only a limited number
of bacterial species are responsible for beer spoilage
and that only a few species are major beer spoilage
bacteria. For quality assurance of finished beer it is
usually practically sufficient to control potential con-
taminations by Lactobacillus brevis, Lb. lindneri,
Pediococcus damnosus, and Pectinatus spp. Most
studies on beer spoilage bacteria have focused on
the taxonomical classification of these bacteria. These
studies have made it possible to identify the bacteria
detected in beer and breweries and to take the proper
measures to control them.
Along with the taxonomical studies, a number of
selective culture media for beer spoilage bacteria have
been developed and recommended by various brew-
ery organizations as shown in Table 2. European
Brewery Convention recommends three media for
the detection of lactobacilli and pediococci: MRS
(de Man, Rogosa and Sharpe) agar supplemented with
cycloheximide to prevent growth of aerobes such as
yeasts and moulds, Raka-Ray medium supplemented
with cycloheximide and VLB S7-S (Versuchs- und
Lehranstalt für Brauerei in Berlin). Other optional
media are UBA (Universal Beer Agar) supplemented
with cycloheximide, HLP (Hsu’s Lactobacillus and
Pediococcus medium), NBB (Nachweismedium für
bierschädliche Bakterien), WLD (Wallerstein Labora-
tory Differential Medium), Nakagawa medium, SDA
(Schwarz Differential Agar) and MRS modified by
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124 111

Table 2 American Society of Brewing Chemists recommends

Examples of culture media for the detection of beer spoilage UBA and Brewer’s Tomato Juice Medium for general
bacteria
microbial detection and other media including LMDA
Media Bacteria Recommended bya
(Lee’s Multi-Differential Agar), Raka-Ray, BMB
b
MRS (de Man, LAB EBC, ASBC, BCOJ (Barney – Miller Brewery Medium) and MRS for the
Rogosa and Sharpe)
detection of lactic acid bacteria and SMMP (Selective
Raka-Ray LAB, G( )c EBC, ASBC, BCOJ
VLB S7-S (Versuchs- LAB EBC, BCOJ Medium for Megasphera and Pectinatus) (The Tech-
und Lehranstalt für nical Committee and the Editorial Committee of the
Brauerei in Berlin) American Society of Brewing Chemists, 1992). Brew-
HLP (Hsu’s Lactobacillus LAB EBC, BCOJ ery Convention of Japan also recommends the use of
and Pediococcus
some of those media (Brewery Convention of Japan,
medium)
WLD (Wallerstein LAB EBC, BCOJ 1999). These culture media are listed in Table 2.
Differential) The conventional detection method based on cul-
Nakagawa LAB EBC, BCOJ turing of the organisms in these media has the
SDA (Schwarz LAB EBC, BCOJ significant disadvantage that is very time-consuming.
Differential Agar)
One week or even longer is needed to obtain visible
Concentrated MRS G( ) EBC, BCOJ
PYF (Peptone, Yeasts G( ) EBC, BCOJ colonies on plates or turbidity in broths. Consequent-
extract and Fructose) ly, the products are often already released for sale
Thioglycolate Medium G( ) EBC before the microbiological results become available.
LL-Agar G( ) EBC, BCOJ Another problem is that these media are not species-
UBA (Universal Beer Agar) LAB, G( ) EBC, ASBC, BCOJ
specific. Media for the detection of beer spoilage
NBB (Nachweismedium für LAB, G( ) EBC, BCOJ
bierschädliche Bakteriën) lactic acid bacteria allow also growth of nonbeer-
Brewer’s Tomato LAB, G( ) ASBC spoilage species such as Lactobacillus delbrueckii
Juice Medium and P. acidilactici. If the selectivity is increased by
LMDA (Lee’s Multi- LAB ASBC the addition of specific chemicals to these media, even
Differential Agar)
longer detection times might be required.
BMB (Barney – Miller LAB ASBC
Brewery Medium)
SMMP (Selective Medium G( ) ASBC, BCOJ 3.2. Identification methods
for Megasphaera and
Pectinatus) Following the bacterial detection in these media,
a
EBC, European Brewery Convention; ASBC, American species identification is needed. Besides the basic tests
Society of Brewing Chemists; BCOJ, Brewery Convention of such as colony morphology, cell morphology, Gram-
Japan.
b staining and catalase assays, also biochemical tests
LAB, lactic acid bacteria.
c
G( ), Gram-negative bacteria. such as sugar fermentation pattern and chromato-
graphic analysis of organic acids can be performed.
Also, specific detection and identification methods are
addition of maltose and yeast extract at pH 4.7. None used such as immunoassays with polyclonal or mono-
of these media are suitable for detecting all strains of clonal antibodies (Claussen et al., 1975, 1981; Dolezil
lactobacilli and pediococci but a combination of some and Kirsop, 1976; Haikara, 1983; Gares et al., 1993;
of these media yields the best results. For the detec- Sato et al., 1994; Whiting et al., 1992, 1999a,b; Ziola
tion of Pectinatus and Megasphaera, the following et al., 1999, 2000a,b), DNA – DNA hybridization,
media are recommended: Concentrated MRS broth, DNA sequencing (Doyle et al., 1995) and polymerase
PYF (Peptone, Yeast extract and Fructose) and Thio- chain reaction (PCR) (Tsuchiya et al., 1992, 1993,
glycolate Medium for enrichment of beer, LL-Agar 1994; DiMichele and Lewis, 1993; Thompson et al.,
for growth in Lee tube, and UBA, NBB and Raka– 1994; Vogesser et al., 1955a,b; Yasui, 1995; Stewart
Ray for routine analysis at breweries. For Zymomonas and Dowhanick, 1996; Yasui et al., 1997; Sakamoto,
spp., Zymomonas Enrichment Medium is also recom- 1997; Sakamoto et al., 1997; Satokari et al., 1997,
mended (European Brewery Convention, 1992). 1998; Juvonen and Satokari, 1999; Motoyama and
112 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
Ogata, 2000; Bischoff et al., 2001). These modern
methods have been reviewed (Barney and Kot, 1992,
Schmidt, 1992; Dowhanick and Russell, 1993; Dow-
hanick, 1995; Schofield, 1995; Hammond, 1996;
Schmidt, 1999).
3.3. Discrimination of beer spoilage bacteria from
nonspoilers
After detection and identification it is for some
species necessary to identify the bacterium as an
actual beer spoiler. While all strains belonging to
Pectinatus spp. and M. cerevisiae have been reported
to be capable of spoiling beer (Haikara, 1991), lactic
acid bacteria include both beer spoilage and non-
spoilage strains. Among Lb. brevis and P. damnosus,
most of the strains are capable of spoiling beer and
only a few strains are not. On the other hand, the
number of beer spoilage strains in Lb. casei, Lb.
coryneformis and Lb. plantarum is limited. Excep-
tionally, all strains of Lb. lindneri have been reported
to be capable of spoiling beer (Rinck and Wackerba-
uer, 1987a,b; Storgårds et al., 1998).
Before the beginning of 1990s, the only method
available for judging the beer spoiling potential of a
bacterium was the so-called ‘forcing test’. In this test,
the bacterium was re-inoculated into beer or beer
enriched with concentrated nutrient medium. Howev-
er, this test has proven to be far from practical for
quality assurance since a few months are needed to
obtain conclusive results.
More rapid procedures have been developed. The
identification at the strain level can now be done at the
genome level. Ribotyping, based on Southern hybrid-
ization with a ribosomal gene as a probe, has been
successfully introduced (Motoyama et al., 1998, 2000;
Satokari et al., 2000; Suihko and Haikara, 2000;
Barney et al., 2001). Fully automated ribotyping
machines now commercially available and only 8 h
are needed to obtain conclusive results. Amplified
Fragment Length Polymorphisms (AFLP) (Perpete et
al., 2001) and Random Amplified Polymorphic DNA
(RAPD-PCR) (Savard et al., 1994; Tompkins et al.,
1996) have also successfully been applied for bacte-
rial strain identification as well as for identification
of brewing yeasts. In these methods, the genotype of
each beer spoilage strain is registered in a database.
A comparison of the genotype of a newly detected
strain with registered genotypes can provide impor-
tant intervention.
Another approach is to determine the common
physiological properties responsible for beer-spoiling
ability. For beer spoilage lactic acid bacteria the
common physiological denominator is hop resistance,
which allows growth of these bacteria in beer. How-
ever, measuring hop resistance by culturing in hop
containing medium, is time-consuming.
4. Antibacterial activity of hop compounds
4.1. History of hop usage in beer
The comfortable bitterness experienced in beer
drinking is characteristic for and is mainly caused
by hop compounds. These hop compounds are present
in the flowers of the hop plant, Humulus lupulus, L.,
which are added to the wort. This plant has been
known for thousands of years. However, its use in
beer is not old as the history of beer itself (5000 – 7000
years). A few descriptions of hops as a beer additive
as well as a decoration for gardens were found in
documents of the 6th century BC. German monks in
the 12th century often used hops in beer making. In
those days, as in ancient times, it was popular to use a
variety of fruits, herbs and spices to flavor beer (so-
called gruit beer). Initially, the bitter taste from hops
was not particularly appreciated. However, when in
the 14th century beer production increased and beer
was exported, the importance of hops in beer was
gradually more and more appreciated, not only for its
contribution to beer flavor but also for its contribution
to the stability. Hopped beer can be preserved signif-
icantly longer than gruit beer. In 1516, Wilhelm IV,
the lord of Bayern, enacted the ‘Reinheitsgebot (Pu-
rity Law)’ which ordered that beer must be made from
barley, water and hops. Since then, the use of hops
became more popular and standard. Many aspects of
this law were adopted by other countries, which made
hops indispensable for the brewing industry.
4.2. Hop plant
The hop plant is a vine, belonging to the family of
hemp. It is dioecious and blooms yearly. Nowadays,
it is mainly cultured for the brewing industry. Only
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
the female flowers, the so-called cones, are used for
beer. The mature cones contain golden resinous
granules, the lupulin, which are the most important
part of the flower for the bitterness and preservation
of beer. Hop resins are extracted and fractionated as
shown in Fig. 1.
4.3. Antibacterial compounds in hops
Hop chemistry has been developed since 19th
century and has been extensively reviewed (Verzele,
1986; Moir, 2000). Research has especially been
focused on the antibacterial properties of hop com-
pounds and the bitter substances derived from hops.
This research goes back to 1888 when Hayduck
showed first that antiseptic properties of the hops
are due to the soft resins (Hayduck, 1888). In the
Institute of Brewing of the United Kingdom, Walker
conducted from 1922 till 1941 a long-term investiga-
tion on ‘the preservative principles of hops’ (Pyman et
al., 1922; Walker, 1923a,b, 1924a,b, 1925, 1938,
1941; Hasting et al., 1926; Hastings and Walker,
1928a,b, 1929; Walker and Hastings, 1931, 1933a,b;
Walker et al., 1931, 1932, 1935, 1940; Walker and
Parker, 1936, 1937, 1938, 1940a,b). The study fo-
cused on the antiseptic properties of a-acids fraction
(humulone) and h-acid fraction (lupulone).
The a-acid fraction is a mixture of homologous
compounds, the a-acids, which are not transferred as
such to beer. During the wort boiling stage in the
brewing process, a-acids are converted by a rear-
Fig. 1. Fractionation of hop resins (Hough et al., 1971).
113
rangement or isomerization to iso-a-acids, which are
much more soluble and bitterer than the original
compounds. This conversion, which is very important
in hop chemistry, was advanced first in 1888 (Hay-
duck). Wieland et al. (1925) suggested that the hy-
drolysis of humulone to humulinic acid proceeded via
an intermediate. Windisch et al. (1927) investigated
the humulone boiling products under alkaline condi-
tions and isolated a resinous and bitter oil termed
‘‘Resin A’’ with chemical properties similar to the
intermediate and isomeric with humulone. Around
1950, Rigby and Bethune, (1952, 1953) showed that
a-acid fraction is a mixture of three major com-
pounds: humulone, cohumulone and adhumulone
(Fig. 2). The bittering compounds of beer were found
to comprise three major analogues of these three a-
acids, which are now known as iso-a-acids: isohumu-
lone, isocohumulone and isoadhumulone (Fig. 2).
Stereoisomers (cis- and trans-) exist for each iso-a-
acid. Finally, the chemical structure and configuration
of naturally occuring ( )-humulone (De Keukeleire
and Verzele, 1970) and isohumolones (De Keukeleire
and Verzele, 1971) were elucidated. The isomerization
yield of a-acids during wort boiling process is low
(typically of the order of 30%) (Hughes, 2000) due to
relatively acidic condition of wort (ca. pH 5.2) and the
adsorption to the wort coagulum during boiling and
fermentation.
h-acids or lupulones in hops are very poorly
soluble in wort and beer and cannot undergo the same
isomerization processes as a-acids. Consequently,
114 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124

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).

they are not transferred to beer and have no direct
value in brewing.
4.4. Antibacterial mechanism of hop compounds
The antibacterial activities of a-acid (humulone)
and h-acid (lupulone) have been studied before 1950.
Their antibacterial activities are higher than of iso-a-
acids but they dissolve to less extent in beer and water.
Studies of the antiseptic properties of hopped wort and
hop boiling product showed that they inhibit growth
of Gram-positive bacteria but not of Gram-negative
bacteria (Shimwell, 1937a; Walker and Blakebrough,
1952). It was first reported by Shimwell (1937a) that
the antiseptic potency of hop increases at lower pH.
Interestingly, he predicted that the antiseptic potency
of hop is associated with permeability changes of the
bacterial cell wall Shimwell, (1937b). The ‘bacterio-
static power’ was also studied of hop compounds,
including humulone and the humulone boiling prod-
uct (Walker and Blakebrough, 1952). The humulone
boiling product had less bacteriostatic potency in malt
extract (pH 5.5) and wort (ph 5.2) than the original
humulone, while its potency was the same at pH 4.3,
the pH of beer. The hop constituents (lupulone,
humulone, isohumulone and humulinic acid) were
found to cause leakage of the cytoplasmic membrane
of Bacillus subtilis, resulting in the inhibition of active
transport of sugar and amino acids (Teuber and
Schmalreck, 1973). Subsequently, inhibition of respi-
ration and synthesis of protein, RNA and DNA was
also observed. Since the iso-a-acids are mainly pres-
ent in beer among the hop resins and their derivatives,
a precise investigation of the antibacterial activity of
iso-a-acids was needed for understanding the preser-
vation or bacterial stability of beer. The molecular
mechanism of antibacterial activity of iso-a-acids and
the effects of pH of the growth medium and other
variables on the antibacterial activity of hop com-
pounds were investigated after 1990 (Simpson and
Smith, 1992). Hop compounds are weak acids and the
undissociated forms are mainly responsible for inhi-
bition of bacterial growth (Fig. 3).
In Lb. brevis (Simpson, 1993b), trans-isohumulone
reduces the uptake of leucine and causes slow leakage
of accumulated leucine. trans-Isohumulone dissipates
effectively the transmembrane pH gradient (DpH) of
the proton motive force but not the transmembrane
electrical potential (Dw). Inhibition of H+-ATPase
activity was not observed. Potentiometric studies
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
Fig. 3. Dissociation of trans-isohumulone (Fernandez and Simpson,
1995b).
revealed that undissociated trans-isohumulone acts
most likely as an ionophore, catalyzing electroneutral
influx of undissociated isohumulone, internal dissoci-
ation of (H+)-isohumulone and efflux of the complex
of isohumulone with divalent cations such as Mn2 +.
This cation is known to be present at high concen-
trations in lactobacillus cells (Archibald and Frido-
vich, 1981a,b; Archibald and Duong, 1984). The
results of this activity is a decrease of the pH gradient
across the membrane. It was reported that the anti-
bacterial activity of trans-isohumulone can be influ-
enced by the presence of cations in the medium.
Protonophoric activity of trans-isohumulone requires
the presence of monovalent cations such as K+, Na+ or
Rb+ and increases with the concentration of these
monovalent cations (Simpson and Smith, 1992).
trans-Isohumulone cannot bind K+ unless a divalent
cation, such as Mn2 +, Mg2 +, Ni2 + and Ca2 + or a
trivalent cation, such as Li3 + and Al3 +, is present in
the medium (Simpson et al., 1993; Simpson and
Hughes, 1993). Thus, the ability of hop compounds
to bind simultaneously two or more cations may be
crucial for their antibacterial action but the reason has
been still unclear.
The properties of other hop acids are similar to
those of trans-isohumulone and it is likely that the
mechanism of their antibacterial activities is also
similar. Some strains of lactic acid bacteria, which
are sensitive to trans-isohumulone, are also sensitive
to ( )-humulone and colupulone and other strains
resistant to trans-isohumulone are also resistant to the
related compounds (Fernandez and Simpson, 1993).
The antibacterial activities of six naturally occurring
iso-a-acids, five chemically reduced iso-a-acids and a
reduced iso-a-acids mixture were higher at lower pH
values while several hydrophobic reduced iso-a-acids
were found to be far more antibacterial than their
115
naturally occurring analogues (Price and Stapely,
2001).
5. Hop resistance in lactic acid bacteria
Beer spoiling lactic acid bacteria have to be hop
resistant in order to grow in beer. The understanding
and elucidation of the mechanism of hop resistance is
not only of scientific interest but is also important for
the microbial control in brewing industry to predict
the beer-spoiling ability of lactic acid bacteria.
5.1. Variation of hop resistance
The extent of hop resistance varies between bacte-
ria. Among beer-spoiling lactic acid bacteria, Lb.
brevis is so far the most resistant to hop compounds.
The degree of hop resistance varies among the differ-
ent strains of Lb. brevis (Hough et al., 1957; Harris
and Watson, 1960; Simpson and Fernandez, 1992;
Fernandez and Simpson, 1993). Hop resistance of
lactobacilli decreases upon prolonged serial subcultur-
ing in the absence of hop compounds (Shimwell,
1936; Yamamoto, 1958; Richards and Macrae,
1964). Hop resistance was thought to be caused by
immunity acquired by prolonged contact with hop
compounds under brewing conditions (Shimwell,
1937a). The necessity of beer spoiling lactic acid
bacteria to acclimatize to beer or hop compounds in
order to reproduce in beer (Yamamoto, 1958) was
solidly documented by Richards and Macrae in 1964.
Hop resistance increased 8- to 20-fold in strains of
lactobacilli upon serial subculturing in media contain-
ing increasing concentrations of hop compounds,
while subculturing of resistant populations in the
absence of hop compounds resulted gradually in
decreased hop resistance. It took about 1 year of
subculturing in unhopped beer to maximally reduce
hop resistance of lactobacilli, indicating that the
acquired hop resistance can be a very stable property
(Shimwell, 1936). However, organisms isolated from
spoiled beer frequently fail to grow upon reinocula-
tion in beer. Preculturing in the presence of subinhib-
itory concentrations of isohumulone is needed in order
to make growth in beer possible (Simpson and Fer-
nandez, 1992). The stability of hop resistance in Lb.
brevis appears to vary from strain to strain. The hop
116 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
resistance in Lb. brevis strain BSO310 could not be
altered by plasmid curing or mutation induced with
ultraviolet light (Fernandez and Simpson, 1993), sug-
gesting that it may be generally a stable character,
both phenotypically and genetically. The Lb. brevis
strain ABBC45 can develop hop resistance in the
same way as observed by Richards and Macrea (Sami
et al., 1997a). Hop resistance increased with the copy
number of plasmid pRH45. When Lb. brevis ABBC45
was cured from this plasmid by serial subculturing in
the absence of hop compounds, the degree of hop
resistance decreased (Sami et al., 1998; Suzuki et al.,
2002). This plasmid contains the horA gene that codes
for a polypeptide that is 53% identical to LmrA, the
lactococcal ATP-binding cassette (ABC) multidrug
transporter (van Veen et al., 1996). HorA protein
was expressed heterologously in Lactococcus lactis
and found to function as an ABC-type multidrug
transporter and to excrete hop compounds (Sakamoto
et al., 2001).
5.2. Features of hop resistance
The pattern of resistance or sensitivity of several
species of lactobacilli to isohumulone is similar to that
of the related hop acids humulone and lupulone
(Richards and Macrae, 1964; Fernandez and Simpson,
1993). Fernandez and Simpson (1993) compared the
properties of hop-resistant strains of lactobacilli and
pediococci with those of sensitive strains. No obvious
correlation was found between hop resistance and cell
morphology, colony morphology, pH range for
growth, sugar utilization profile, products of metabo-
lism, manganese requirement and sensitivity to super-
oxide radicals, expression of cellular proteins and
resistance to various antibacterial agents. However,
differences were found in the transmembrane pH
gradient (DpH) and the cellular ATP pool. Because
hop compounds act as protonophores that dissipate
the DpH across the cellular membrane (Simpson,
1993a,b), these differences are of great importance
for understanding the mechanism of hop resistance.
5.3. Mechanisms of hop resistance
The molecular structure of antibacterial agents
might supply an insight in the mechanism of resis-
tance. Resistance to trans-isohumulone also results in
resistance to ( )-humulone and colupulone (Fernan-
dez and Simpson, 1993), suggesting a common mech-
anism 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, van-
comycin) (Fernandez and Simpson, 1993). The resis-
tance 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 be-
ta-lactam ring into innocuous substrates. In hop-resis-
tant strains of Lb. brevis, neither conversion nor
inactivation of trans-isohumulone was found (Simp-
son 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 hop-
resistant Lb. brevis has also a changed lipid compo-
sition 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 Gram-
positive mycobacteria has been found to be an excep-
tionally efficient barrier. The permeation of hop com-
pounds 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
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124 117

membrane of many bacteria has been extensively
documented (Putman et al., 2000). A multidrug resis-
tance pump HorA (Sakamoto et al., 2001) has been
found in Lb. brevis ABBC45, which is overexpressed
when exposed to hop compounds (Sami, 1999). In
addition, a proton motive force-dependent hop excre-
tion transporter was suggested in this strain (Suzuki
et al., 2002).
(v) other mechanisms to tolerate the toxic effects of
drugs. Since hop compounds act as protonophores and
dissipate the transmembrane pH gradient (DpH), the
cells could respond by increasing the rate at which
protons are expelled. The hop-resistant strains main-
tain a larger DpH than hop-sensitive strains (Simpson
and Fernandez, 1993) and Lb. brevis ABBC45
increases its H+-ATPase activity upon acclimatization
to hop compounds (Sakamoto et al., 2002). The ATP
pool in hop-resistant strains was also found to be a
larger than in hop-sensitive strains (Simpson and
Fernandez, 1994; Okazaki et al., 1997). The ability
of hop-resistant strains to produce large amounts of
ATP in the cell is needed for the increased activity of
the H+-ATPase and for the hop extruding activity of
HorA. It is interesting that these responses occurred
also in hop-sensitive strains at subinhibitory concen-
tration of trans-isohumulone (Simpson, 1993a; Simp-
son and Fernandez, 1994). However, at higher
concentrations, both the DpH and the pool decreased
in the hop-sensitive strains, but not in the hop-resis-
tant strains. A role of HitA in hop resistance was
suggested (Hayashi et al., 2001). It is about 30%
identical to the natural resistance-associated macro-
phage proteins (Nramp), which function as divalent-
cation transporters in many prokaryotic and eukary-
otic organisms. HitA could play a role in transport of
divalent cations, while isohumulone has been claimed
to exchange protons for cellar divalent cations such as
Mn2 +. Thus, a simple mechanism of hop resistance
does not appear to exist. The resistance mechanisms
found so far in Lb. brevis are illustrated in Fig. 4.

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 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
6. Beer-spoiling ability in lactic acid bacteria
Hop resistance is crucial to beer-spoiling ability of
lactic acid bacteria. In many bacteria, hop-resistance
mechanisms need to be induced before growth in beer
is possible. For this induction, some bacteria need to
be exposed to subinhibitory concentrations of hop
compounds (Simpson, 1993a,b).
6.1. Factors affecting the growth in beer
Beer spoilage and bacterial growth depend on the
strain and the type of beer. The ability of 14 hop-
resistant lactic acid bacterial strains, including Lb.
brevis and P. damnosus strains, was investigated for
their capacity to grow in 17 different lager beers with
the biological challenge test (Fernandez and Simpson,
1995a). A statistical analysis of the relationship be-
tween spoilage potential and 56 parameters of beer
composition revealed a correlation with eight parame-
ters: pH, beer color, the content of free amino nitrogen,
total soluble nitrogen content and the concentrations of
a range of individual amino acids, maltotriose, undis-
sociated SO2 and hop compounds. The effects of
dissolved carbon dioxide (CO2) and phenolic com-
pounds including catechin, gallic, phytic and ferulic
acids on beer spoilage were also investigated (Ham-
mond et al., 1999). CO2 was found to inhibit the
growth of lactobacilli at the concentrations present in
typical beer but stimulate at the lower concentrations.
Among the phenolic compounds, ferulic acid, a com-
ponent of barley cell wall and hence present in all
beers, exerted a stronger antibacterial activity after
enzymatic conversion into 4-vinyl guaiacol. Organic
acids in beer may also influence bacterial growth but
this aspect has hardly been studied.
6.2. Prediction of beer spoilage by lactic acid
bacteria
A number of attempts have been made to develop
methods to predict beer-spoiling ability. For lactic
acid bacteria, hop resistance is the key factor. As
described above, some factors have been identified
to cause hop resistance. Rapid procedures for detect-
ing these factors would be very beneficial for micro-
bial control in breweries. A set of PCR primers have
been made that can specifically detect the horA gene
or its homologues in a wide range of lactobacilli
(Sami et al., 1997b). Most horA-positive strains were
found to have beer-spoiling activity, indicating that
this is a very useful prediction method. Another
prediction method is based on ATP pool measure-
ments in lactobacillus cells (Okazaki et al., 1997).
Polyclonal and monoclonal antibodies specific on-
ly for beer spoilage strains have been reported. A
series of antisera were made against the Lactobacillus
group E antigen, a cell wall glycerol teichoic acid
beneath the S-layer protein (Yasui et al., 1992, 1995;
Yasui and Yoda, 1997a) and known to be present in
Lb. brevis, Lb. buchneri, Lb. delbrueckii subsp. lactis
and subsp. bulgaricus (Sharpe, 1955; Sharpe et al.,
1964). When the beer spoilage strain Lb. brevis 578
was used as an antigen, the resulting antiserum
reacted specifically with other beer spoilage strains
of Lb. brevis. Surprisingly, this antiserum also reacted
with beer spoilage strains of P. damnosus, but not with
any strains of Lb. linderi (Yasui and Yoda, 1997a).
Galactosylated glycerol teichoic acid was found to be
the most likely epitope that presumably selectively
increases the cell barrier to hop compounds (Yasui
and Yoda, 1997b). Three monoclonal antibodies spe-
cific for beer spoilage ability of lactic acid bacteria
were obtained by immunizing mice with cells cultured
in beer (Tsuchiya et al., 2000). The monoclonal
antibody raised against Lb. brevis reacted with all
beer spoilage strains of Lb. brevis and several beer
spoilage strains of P. damnosus, but not with non-
spoilage strains of Lb. brevis, P. damnosus and other
lactic acid bacteria. The monoclonal antibody raised
against P. damnosus reacted significantly with all beer
spoilage strains of P. damnosus and weakly with many
of the beer spoilage strains of Lb. brevis. On the other
hand, the monoclonal antibody raised against Lb.
lindneri reacted specifically only with Lb. lindneri.
The reactivity of the still unknown antigens did not
change regardless of the presence of hop compounds
in their culture media.
D-lactate dehydrogenase (LDH) of 60 strains of Lb.
brevis, including 44 beer spoiling strains and 16
nonspoiling strains, was also investigated. The strains
could be divided in five groups (A, B, C, D and E) on
the basis of the mobility of their D-LDH in native
polyacrylamide gels (Takahashi et al., 1999). Forty
out of forty-four beer spoilage strains were classified
to the group B, suggesting a relationship between the
 K. Sakamoto, W.N. Konings / International Journal of Food Microbiology 89 (2003) 105–124
D-LDH profile and beer-spoiling ability. The purified
D-LDH of those groups had different pH and temper-
ature optima and isoelectric points. Especially the
temperature optimum of 50 jC of the Group B D-
LDH is significantly lower than that of the other D-
LDHs (60 jC).
Except for the forcing test, none of the other
methods so far available can predict beer-spoiling
ability of all strains. Since hop resistance in lactic
acid bacteria does not appear to be a general feature of
all beer-spoiling bacteria, combination of several
methods will be required to detect all potential beer
spoiling strains.
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