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tmi Journal

offbodMicrobii
International Journal of
Food Microbiology 33 (1996) 139-155

Specific spoilage organisms in breweries and


laboratory media for their detection

L. Jespersen, M. Jakobsen
The Royul Veterinary and Agricultural University, Depurtment of Dcriry und Food Science, Food
Microhioiogy, Rolighedswj 30, DK-1958 Frederiksberg, Denmark

Abstract

The Gram positive bacteria are generally regarded as the most hazardous beer spoilage
organisms in modern breweries, especially the lactobacilli: L. bveois, L. lindneri, L. ~~rvatu.~,
L. casei, L. huchneri, L. coryne$wmis, L. plantarum, L. hrevisimilis, L. mukffirmentans and L.
parabuchneri and the pediococci: P. damnosus, P. inopinatus and P. dextrinicus. Micrococcus
~r~stin~~e is the only species within the micrococci relevant to brewing. The Gram negative
strictly anaerobic bacteria are apparently increasing in importance and include Pectinatus
c~~rev~.~i~phiiu.~,
Pecfii~atus frisinge~zs~s and Se~eno~nonus lactic{feu, reported as obligate beer
spoilage organisms; Zyt~l~p~~ilu.~ra~~nus~vora~~sas a potential beer spoilage organism: Mexus-
p~zaera cerevisiae as an obligate spoilage organism of low alcohol beer and Zyrnai?7arzus
~~~obi~isas capable of spoiling primed beer. With improved process technology the impor-
tance of aerobic bacteria has decreased and the same applies for the Gram negative aerobic
bacteria Hufiia protea and Enterabacter clvacae which are capable of surviving beer
fermentation.
ljeer spoilage organisms include several so-called wild yeasts, of which Saccharomyces
species are generally considered the most important.
Even though the detection of beer spoilage organisms by cultivation in laboratory media
does not always provide the specificity and the sensitivity required, the use of selective media
and incubation conditions still appears to be the method preferred by breweries. The media
used depend on the type of sample, the specificity required and, for detection of wild yeasts,
to some extent, the characteristics of the culture yeast. Among the media reported so far no
single medium can be used to detect all members within a group of specific beer spoilage
organisms and further work on the development of improved substrates arc required both
for bacteria and wild yeasts.

* Corresponding author. Tel.: + 45 35 283266; fax: + 45 35 283214; e-mail: Ij@kvl.dk

0168-l605/96j$lS.00 0 1996 Eisevier Science B.V. All rights reserved


PiI SOl68-1605(96)01154-3
Kr~~ord~~ls:Beer; Media: Specific spoilage organisms; Lactic acid bacteria; Yeasts

1. Introduction

In a brewery specific spoilage organisms may be defined as any organism which


is not deliberately introduced and which is able to survive and proliferate in the
environment i.e., in wort, fermenting wort, beer after filtration or in packaged beer.
Many bacterial strains have been shown to retain their viability for extended
periods in beer but as long as they are not able to grow in the product they are not
harmful and should not be considered as beer spoilage organisms (Lawrence and
Priest, 1981). With the improved technology introduced in modern breweries the
importance of various specific spoilage organisms has changed. Well known
spoilage organisms like the aerobic acetic acid bacteria i.e. G~~~~?l~~~~~i~~ctq~krns
and A~~~f~~~~~t~~spp. appear no longer to be a problem. Improvements in beer
handling and bottling technology have resulted in significant reduction of the
oxygen content during the process and in packaged beer wjhich has permitted the
growth of strictly anaerobic microorganisms. Typical examples are P~~ti~z~~tu,sspp.
and ~Jqtrs~&~rrzl wrwisiae which within the past few decades have caused increas-
ing problems with spoilage of packaged beer (Lee, 1994).
As mentioned, oxygen content is a major factor in controlling the microflora
capable of growth during beer production and storage. However, several other
factors are also important for the spoilage potential of beer and include the pH
(3.8-4.7) the concentration of hop bitters (approx. 17-55 mg iso-z-acids/l),
ethanol (O-8% w/w), CO, (approx. 0.5% w/v), SO, (approx. 5-30 mg/l), organic
acids, acetaldehyde and other metabolites as well as the nutrient contents and
storage temperature (Hough et al., 1982). The sensitivity of different brands of beer
to spoilage by hop-resistant bacteria belonging to the genera Lc~ctohtrcilkrs and
Prdiococws has been investigated by Fernandez and Simpson (1995) who were able
to predict the spoilage potential of these bacteria for beer from information on the
level of undissociated SO,, undissociated hop bitter acids, polyphenols. maltotriose
and free amino nitrogen as well as colour intensity.
The detection of beer spoilage organisms is difficult as they are often present in
low numbers e.g. for wild yeasts the methods used should be capable of detection
of one spoilage organism per IO culture yeast cells (Thurston, 1986; de Angelo and
Siebert, 1987). Also spoilage organisms are often sublethally damaged due to the
environment~~l conditions. In addition some organisms may be very fastidious
concerning their conditions of growth (Lu~t~~~~~~~~~t~~ spp. and P~~~~~~~~r~.~spp. ) or
may be adapted to the particular product or environmeIlt and very reluctant to
multiply in other environments including highly nutritious laboratory media. For
the detection of wild yeasts the fact that contaminants belonging to the genus
Smdzurornycrs are often biochemically and physiologically very similar to the
culture yeast is a major problem.
L.. Jespersen, M. Jakobsen /ht. J. Food Microbiology 33 (1996) 139-155 141

Within the past twenty years considerable interest has been shown in the
development of rapid methods for detection of beer spoilage organisms. These
methods include bioluminescence techniques, direct epi-fluorescence filter tech-
niques (DEFT), immunoassays, use of automated turbidimetry, measurement of
impedance or conductance, flow cytometry as well as a number of methods
involving DNA technologies such as PCR and DNA hybridization techniques
(Jakobsen and Lillie, 1984; Navarro et al., 1987; Miller and Galston, 1989: Haikara
et al., 1990; Vogel and Bohak, 1990; Hutter, 1991; Barney and Kot, 1992;
DiMichele and Lewis, 1993; Jespersen et al., 1993). With the exception of the
bioluminescence technique which has been used successfully for control of cleaning
and disinfection as we11 as for last rinse water from cleaning in place (CIP), these
alternative methods seem not to have found their way into the breweries as they
often lack the speed, sensitivity and specificity required or include the use of
advanced, expensive equipment and reagents. For these reasons the use of selective
media and incubation conditions is still the method preferred in most breweries.
However, due to the diversity of the microflora, several media must be used in
order to ensure the detection of both Gram positive and Gram negative spoilage
bacteria as well as Succhuromyces and non-Succharomyces wild yeasts.
This review presents a summary of the specific spoilage organisms important to
modern breweries and the selective media and incubation conditions relevant to the
detection of these organisms.

2. Specific spoilage organisms

2.1. Gram positive bacteria

The bacteria generally regarded as most hazardous for modern breweries are the
Gram positive bacteria belonging to the genera Lactobacillus and Pediococcus.
Among the lactobacilli the most important spoilage organisms according to the
brewing literature are Lactobacillus breois, L. lindneri, L. curautus, L. cusei, L.
buchneri, L. corynejtirmis and L. phnturum (Priest, 1996). In addition, the following
potential beer spoilage species have been reported: L. breuisimilis (Back, 1987) L.
mul&rmentans and L. purabuchneri (Farrow et al., 1988). L. delbrueckii, L.
j&wentum and L. j&ctivormns have been reported to occur in beer but their
spoilage potential is low (Priest, 1996). Not all lactobacilli reported seem to be
recognized as valid species according to recent reviews of the genus (Hammes and
Vogel, 1995; Schleifer and Ludwig, 1995) and the brewing literature in general
appears taxonomically out of date.
Among the pediococci only Pediococcus dumnosus, P. inopjn~tz~s and to some
extent P. dextrinicus are of importance for spoilage of beer. However, growth of P.
inop~natus and P. de~~tr~~ieus is only possible above pH 4.2 and at low ethanol and
hop bitter concentrations (Lawrence, 1988). It also appears that only some strains
of the above species are capable of growth in beer (Simpson and Taguchi, 1995). P.
acidilactici and P. pentosuceus are found on malt and can grow during the early
stages of wort production as long as the temperature is below 50C and hops have
not been added, but they have never been reported to cause any defect in the beer
produced (Simpson and Taguchi, 1995).
The genus ~i~~~~~c~~t~~ includes one species, iM. ~~~~s~i~~~~~ relevant to breweries. It
is very sensitive to the collcent~tioll of ethanol and hop bitters in beer and only
capable of growth above pH 4.5 (Back, 1981). Unusually for the micrococci, M.
kristinrte is capable of anaerobic growth (Lawrence and Priest, 1981).
The spoilage caused by lactic acid bacteria appears to be dependent on the
composition of the beer produced and to be most dangerous during conditioning of
beer and in packaged products (Priest, 1996). Gram positive bacteria are generally
linked to haze or rope formation and acidification. In particular t. cc~~~i and the
P&&occ~~ spp. produce extensive amounts of diacetyl. Honey-like fiavours and
extended ~erment~~tion time have been linked with infections caused by P~~~~~[~~~(~~,~~.~
spp. and L. hrez~is has been shown to cause super-attenuation due to its ability to
ferment dextrins and starch (Lawrence, 1988). A fruity atypical arotna has been
reported for .M. kri.stinae even when present at low numbers (Back. 1981).
As indicated above, the spoilage potential of lactic acid bacteria is to a large
extent dependent on their resistance to hop bitters such as tvuns-isohumulone. The
effect of hop bitters can be both bacteriostatic and bactericidal depending on their
concentrations and the exposure time, however, the adaptation of cells to hop
bitters has been questioned by Simpson (1993). The antimicrobial activity is related
to the undissociated form of the hop bitter acids and thereby pH dependent, e.g. the
antibacterial activity of ~~~~~.~-isohumulo~e towards L. hr&s decreases LOO-fold if
the pH is raised from 4.0 to 7.0 (Simpson, 1993). It appears that hop bitter acids
act as ionophores, carrying ions including protons across the plasma membrane and
reducing the intracellular pH of the cell. The exact mechanism responsible for the
increased resistance of some strains appears not to be known but might according
to Simpson (1993) and Simpson and Fernandez (1994) be related to the plasma
membrane.

2.2. G~UFR ncgcrtiw bacteria

The Gram negative beer spoilage organisnls include a number of species belong-
ing to various genera. Among these the strictly anaerobic bacteria Pectin~~tu.s
P. .~~~~jng~nsi~ and S~~l~n(~~~~~u~ IrrctkYfxx
~~r~?~~~~~~h~I~,i~s~ have been reported as
obligate beer spoilage organisms (Schleifer et al., 1990; van Vuuren, 1996). Within
the genus ZymopMus which is phylogenetically close to the genus Pectinntus, two
species have been isolated from brewery samples: Z. ~~~~~~~~~i~or~~~ and Z. paucivo-
~UIZS although onIy the first mentioned has been reported as a beer spoilage
organism (Schleifer et al., 1990; Seidel-Refer, 1990). The beer spoilage organisn~s
belonging to the genera P~~~~~uru~ and Z~lrn~~~~~~~ have been reported to grow in
beer at pH above 4.3-4.6 and at ethanol concentrations below 5% (w/v). These
strictly anaerobic bacteria have become more important with the increasing produc-
tion of non-pasteurized and flash pasteurized beer together with improved bottling
technology which results in significantly reduced oxygen content in packaged beers.
The spoilage caused by these organisms includes the production of propionic,
acetic, and succinic acids, methyl mercaptan, dimethyl sulphide and hydrogen
sulphide as well as turbidity (Lawrence, 1988; Seidel-Riifer, 1990).
Another strictly anaerobic Gram negative beer spoilage organism is the coccus
Mega.@z~~era cerevisiue. It is sensitive to low pW ( < 4.1) and growth is inhibited in
beer with an ethanol content above 2.8% (w/v), however, growth has been observed
in beer with up to 3.8% (w/v) ethanol (Haikara and Lounatmaa, 1987; Lawrence,
1988). As for Pectin&us ~erez~i~~~jp~zjl~s it has proved to be quite toierant towards
hop bitters and it may cause spoilage of low alcohol beer by production of butyric
and other fatty acids as well as hydrogen sulphide and development of turbidity
(Haikara and Lounatmaa, 1987; Lee, 1994).
Of importance for pritned (added sugars) beer is the anaerobic but oxygen
tolerant Gram negative bacterium Zymomonas mobilis which is resistant to hop
bitters and able to grow at pH above 3.4 and ethanol concentrations lower than
10% (w/v). It is not able to ferment maltose and maltotriose but it ferments glucose
and fructose, and some strains ferment sucrose. Spoilage of primed beer is primarily
caused by the production of high levels of acetaldehyde and hydrogen sulphide.
Zyt~~o~~onas spp. have not been reported in lager breweries probably due to their
stringent carbohydrate requirements (van Vuuren, 1996).
The Gram negative, aerobic acetic acid bacteria, especially ~luco~~obucter oxy-
dans, Acetobacter aceti and A. puste~ria~l~s have during the history of brewing been
paid a great deal of attention. They are able to convert ethanol into acetic acid and
thereby change the flavour of the beer significantly, resulting in vinegary off-
llavours (Lawrence, 1988). However, being aerobes they are not considered a major
problem in modern breweries. The same applies to the Enterobacteriaceae, among
which a variety of species have been found in breweries including Hufniu protea,
Hafniu alvei, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes
and Enterobacter ugglomerans, now recognized as Rahnella aquatilis (van Vuuren,
1996). These members of the family Enterobacteriaceae are considered as wort
spoilers as they do not multiply in beer (Lawrence, 1988). The two species H. protea
and E. cloucae can survive the fermentation process and since they cosediment
together with the culture yeast may be passed on to subsequent fermentations
(Thastum and Jakobsen, 1981). Comparing the survival of the two species during
yeast storage (lO*C) H. protea has been shown to have a significantly higher
resistance than E. doacae (Thastum and Jakobsen, 1981). They can cause beer
spoilage by production of fuse1 alcohols, 2,3_butanediol, dimethylsulphide (DMS)
and dimethyl disulphide which are transferred to the final beer (Lawrence, 1988).
Table 1 lists the bacteria mentioned in the brewing literature as capable of
growth in beer.

2.3. Yeasts

The diversity of wiid yeasts in terms of beer spoilage means that no general
description can be given, however, wild yeasts are generally divided into Succha-
romyces and non-~ffcchur~~l_~ce~ wild yeasts. Often the most severe infections will
be caused by ~acc~uro~?7~7~esspp. which, once isolated, can often be distin~ished
from lager yeasts by cell morpholo~ and spore formation (Jorgensen, 1948;
Ingledew and Casey, 1982; van der Walt and Yarrow, 1984).
Among the Saccharomyces wild yeasts, most isolates belong to Succharornyccs
ceretisiue with a predominance of strains previously described as Succh. diustnticus,
Succh. pastorianus, Succlz. ellipsoidtxs and Sr~ch. wdliunus (Yarrow, 1984). Infeo-
tions with these yeasts typically cause phenolic off-flavours and super-attenuation
of the final beer. The production of phenolic off-favours is due to the ability of
these wild yeasts to decarboxylate different phenolic acids such as ferulic and
trans-cinnamic acids (Wackerbauer et al., 1977; Ryder et al., 1978; Meaden and
Taylor, 1991) while the super-attenuation is due to the production and secretion of
glycoamylases with starch deb~~nchillg activity which enables the wild yeasts to use
dextrins not normally fermented by the culture yeast (Rocken and Schulte, i986).
The most important non-S~i~~lrurortl~~c~es wild yeasts are Pichitt ,I?~mhrcilzt~~~rfil~i~~.~
and Hurmwul~~ momala as well as a number of species belonging to the genera
Torzrlopsis, Schi~osac~clzarnt~~~t~e.s, %rett~~i70t11!.LtJ~s,
Kloctkera and Cmdiria (Anony-
mous, 1981; Ingledew and Casey, 1982). Among the Ctrndid~~spp. , <. ~~IJxY~~~~I

Table I
Specilic spoilage bacteria capable of growth m beer

* Restricted by high ethanol and hop bitter concentrations


Grows only in low alcohol beer.
c_Grows only in primed beers.
L. Jespersen, M. Jakob.~e~ / ht. J. Food ~~icrobio~~g?33 (1996) 13Sl5.5 145

Table 2
Sensitivity required for detection of specific spoilage organisms in brewery samples

Samples Sensitivity
_.__
Cold aerated wort I organism per 25 ml
Pitching yeast I bacterium per ml and 1 wild yeast per IO6 culture
yeast
Fermenting wort I organism per ml
Tank bottoms I organism per ml
Beer in storage 1 organism per ml
Filtered beer 1 beer spoilage organism per 100 ml or lo- IO2 non-
beer spoilage organisms per 100 ml
Packaged beer (non-pasteurized or flash IO- IO non-beer spoilage organisms per 100 ml
pasteurized)
Rinse water (end of cleaning in place) I organism per 100 ml

and C. krusei have been reported as capable of beer spoilage (Ingledew and Casey,
1982). The non-Succharontyces wild yeasts will cause various types of spoilage, e.g.
Pichia membraneJaciens is known to produce film, haze and off-fhavours and
Torulopsis spp. are known to cause super-attenuation. According to Campbell and
Msongo (1991) spoilage caused by wild yeasts belonging to the genera Pi&a,
Hun.~e~~uia and Deba~yo~~y~es is commonly associated with aerobic conditions even
though the yeast species are capable of anaerobic growth.

3. Media and methods for detection of spoilage organisms

3.1, Microbiological guidelines and sensitivity of methods

The presence of specific spoilage organisms even at very low levels constitutes a
risk considering the extended shelf life (six months or longer) of beer and the
relatively long process time i.e. Z--3 weeks. In practice microbiological guidelines
will relate to the maximum sample size which can be handled and the detection
levels obtainable in routine microbiological analysis. The levels for detection of
specific spoilage organisms required at various stages during the brewing process
are indicated in Table 2. The efficiency of the cleaning and disinfection of
processing and packaging equipment must also be controlled as both Gram positive
and Gram negative spoilage organisms have been shown to accumulate in biofilms
on stainless steel surfaces in breweries (Czechowski and Banner, 1992).
As indicated in Table 2 different analytical sensitivities are required for the
various stages of the brewing process. Cold wort (25550 ml) is examined by a
forcing test including incubation of the wort at 25C for 3-5 days followed by
visual inspection for the presence of microbial growth with subsequent subcultiva-
tion and microscopy as required. For detection of specific spoilage bacteria in
pitching yeast slurries, fermenting wort, tank bottoms and beer in storage, 1 ml
samples are incubated in selected solid or liquid media. For beer after filtration,
packaged beer and last rinse water from CIP, 100 ml samples are membrane fihered
with incubation of the filter on selected media (Anonymous, 1977; Anonymous,
1981; Anonymous, 1992a). For most purposes no single medium offers the specifi-
city and growth conditions required and various media, as described below, have to
be included.
Before describing the media for detection of specific spoilage organisms it should
be mentioned that use of a non-selective medium like standard plate count agar
(PCA) for determination of a mesophilic, aerobic count can serve as a useful
indicator of general hygiene of breweries.

Numerous media have been reported for the detection of lactobacilli and
pediococci. They include standard media for detection of these organisms such as
MRS agar supplemented with maltose, sucrose agar and Raka-Ray lactic acid
bacteria medium (Anonymous, 1981). Other media developed specifically for the
brewing industry are based upon the use of beer as the basic component in addition
to complex nutrient ingredients, minerals and various growth factors. These media
include universal beer agar, UBA (Kozulis and Page, 196X; Anonymous, 1992a),
nachweismedium fiir bierschgdliche bacterien, NBB medium and modified NBB
medium (Nishikawa and Kohgo, 1985; Kindraka, 1987; Anonymous, 1988), VLB-
S7 (Emeis. 1969) and KOT medium (Taguchi et al.. 1990) Compositions of UBA.
modified NBB medium and KOT medium are given in Tables 3-5 respectively. For
these media the ingredients in the beer incfuded will inhibit the growth of most
other bacteria than beer spoilage organisms. In KOT medium actidione is further
added in order to prevent the growth of culture and wild yeasts, and sodium azide
in order to prevent the growth of Gram negative bacteria (Taguchi et al., 1990).
The exact composition of VLB-S7 appears not to have been reported. For media
which incorporate beer, beer from the actual brewery should be used as it is likely
that the beer composition will influence the spoilage organisms capability of
growth. Lactic acid bacteria should be incubated anaerobically or microaerobically
due to the microaerophilic nature of these microorganisms (Matsuzuwa et al.,

Table 3
Compositioll of UBA
~-~.
Tomato juice broth 25.0 g
Peptonired milk 15.0 g
Dextrose 10.0 g
Agar 12.0 g
Distilled water 750 ml
Beer 250 ml

Final pH 6.3
-.
Anonymous, 1992a.
L. Jespcrscn, M. Jakohsen /ht. J. Food Microhiolog)~ 33 (1996) 1399155 147

Table 4
Composition of modified NBB medium

Casein peptone 5.0 g


Yeast extract 5.0 g
Meat extract 2.0 g
Tween-80 0.5 ml
Potassium acetate 6.0 g
Sodium phosphate. dibasic 2.0 g
L-Cysteine monohydrochloride 0.2 g
Chlorophenolred 70 mg
Glucose 15 g
Maltose 15 g
I.-Malic acid 0.5 g
Agar 15 g
Beer/distilled water (1:l), ad 1000 ml

Final pH 5.8

Kindraka, 1987.

1979). Normally a long incubation time is required i.e. 5-7 days and incubation at
28-30X is often used.
No single medium appears to be capable of detecting all strains of tactic acid
bacteria and various results have been obtained by use of these media. For
Raka-Ray medium excessive growth of non-spoilage bacteria has been reported

Table 5
Compositioll of KOT medium

Trypticase peptone 5.0 g


Malt extract 2.5 g
Yeast extract 2.5 g
Liver concentrate I.0 g
Maltose 5.0 g
Glucose 5.0 g
L-Malic acid 0.5 g
Tween-80 I ml
K,HPO, 0.5 g
MgSO,.7H,O 0.125 g
MnSO,,4-5H20 0.025 g
Cyst&e monohydrochloride 0.5 g
Cytidine 0.2 g
Thymidine 0.2 g
Actidione 100 mg
NaN, SO mg
Agar 20 g
Beer 800 ml
Distilled water, ad 1000 ml

Final pH 6.3

Taguchi et al., 1990.


148 L. Jrspursm, M. Jukohsen ! Int. J. Food Microbiology 33 (1996) 139--155

Table 6
Composition of SMMP medium
_--
Yeast extract
Peptone
w-lactic acid sodium salt (60% syrup)
Sodium thio~lycoll~te
L-cysteine HCI
K,HPO,.3H,O
K H,PO,
N&i
(NHJlHPO,
NaC,H,0Z~3H,0
Sodium fusidate
Actidione (cycloheximide)
Crystal violet
Beer
Distilled water, ad
~-
Lee, 1994.

(Matsuzuwa et al., 1979). Wackerbauer and Rinck (1983) found that VLB S7
medium was more selective for beer spoilage organisms than NBB medium on
which Enferohactrr, Pseudomonas. Hajku and Klebsiella spp., and yeasts were
found to grow. However, Back et al. (1984) found that the original NBB medium
and a modified NBB medium gave more rapid results and higher detection rates of
beer spoilage organisms than VLB S7 medium. Kindraka (1987) found that
modified NBB medium was specific and showed higher counts for beer spoilage
organisms than CUBA, but this could not be confirmed in a comparative study
conducted by the American Society of Brewing Chemists (Anonymous, 1958).
Taguchi et al. (1990) found that KOT medium supported better growth of slow
growing lactic acid bacteria than modified NBB or VLB-S7 medium. These
investigatiot~s suggest that the optimal medium for detection of lactic acid bacteria
in brewery samples has not yet been identified.

3.3. Detection of strir.tly unuerobi~ Grcrr31negtltiw bwteriu

For detection of Pectinatus ecrecisiiphitus and other strictly anaerobes pre-re-


duced NBB (Henriksson and Haikara, 1991) or pre-reduced MRS (Gares et al.,
1993) can be used. Megusphaera spp. have been reported to grow on pre-reduced
peptone yeast extract agar with added glucose or fructose (Haikara and Lou-
natmaa, 1987). For the selective detection of Puctinatus spp. and Megasphuercr
cereuisiae, SMMP medium (Selective Medium for Megusphaeru and Pectinutus) has
recently been reported (Lee, 1994). SMMP medium is beer-based and supplemented
with reducing agents, various nutrients, 1.0% lactate as the sole carbon source, 20
ppm actidione (cycloheximide) to inhibit yeasts, and 25 ppm crystal violet and 5
ppm sodium fusidate to inhibit Gram positive bacteria. The exact composition of
the SMMP medium is given in Table 6. The medium was originally prepared as a
L. Jespersen, M. Jakohsen i Int. J. Food Microbiology 33 (1996) 139-155 149

liquid medium, but can be used as a solid medium as well. In this case evaporation
of ethanol from the beer included in the medium may cause loss of selectivity which
can be compensated by addition of ethanol (Lee, 1994). As these strictly anaerobic
bacteria are very oxygen sensitive care should be taken not to expose the samples
to air.

3.4. ~ete~~ti~~ qf other bacteria

Zymmonus spp. in brewery samples can be detected in malt extract, yeast


extract, glucose, peptone agar (MYGP) adjusted to pH 4.0 with lactic acid and
added 20 ppm actidione and 3% ethanol (v/v) or in beer with added sugars and fO0
ppm actidione, incubated anaerobi~dlly at 25530C for 2-6 days (Anonymous,
1981). A Iiquid medium containing glucose, fructose and yeast extract supple-
mented with 100 ppm actidione and adjusted to a pH of 4.0 has also been reported
for detection of Zymonl~na.s spp. (Woodward, 1982). Use of media incorporating
either sulphite or lead acetate for the production of black colonies or Schiffs
reagent for the production of purple colonies has been reported for the specific
detection of Z~~?omonus spp. (Dennis and Young, 1982; Woodward, 1982). Ac-
cording to Woodward (1982) incubation of membrane filters on agar plates is not
satisfactory for detection of Zy~~i~on~s spp. in beer.
Differential media are generally not used for acetic acid bacteria which can be
detected in e.g. PCA incubated aerobically at 28C for 1-3 days (Anonymous,
1981). Acetobucter spp. have been reported to grow well on a medium containing
0.50/;) (w/v) yeast extract, 1.5% ethanol (v/v) and 2.5% (w/v) agar (van Vuuren,
1996). For detection of Enterobacter~ceae in wort and yeast slurries the European
Brewing Convention recommends the use of MacConkey agar with added actidione
(10 ppm) in order to inhibit growth of yeasts and most other microorganisms
present in wort (Anonymous, 1981). Although this medium is recommended in the
brewing literature it may not be optimal considering the cell damage of Enterobac-
teriaceae present in brewery samples.

No single method or medium will allow the detection of all wild yeasts while
suppressing culture yeasts which means that complementary methods and media are
required. A method traditionally used for detection of wild yeasts in pitching yeast
is the sporulation test where the yeast sample on a wet filter paper is exposed to
potassium acetate or sodium acetate either directly or by growth on an acetate
containing medium (Jorgensen, 1948). Acetate induces the sporulation of spore-
forming wild yeasts which by microscopy can be distinguished from culture yeasts
as these have generally either lost this capacity or form spores later than the wild
yeasts. Microscopy and immunofluorescence staining have also been used to detect
wild yeasts present in samples of pitching yeast (Chilver et al., 1978; Kersten and
Zahn, 1988), however, lack of specificity and sensitivity is a limiting factor for these
techniques. The most widespread methods for wild yeast detection still rely upon
the use of various solid media.
Crystal violet medium, Wort Agar (WA) with 10. 20 ppm crystal violet as the
only selective agent, has a long tradition for detection of SU~~~~?U~~~~I~C~~S wild yeasts
(Anonymous, 1981). Unfortunately, a wide range of sensitivities to crystal violet
has been demonstrated among S~rc~h~lrro~~~,~~~~,~culture yeasts as well as $X&I-
RI~?.V~V.S wild yeasts which makes the universal use of the crystal violet medium very
difficult (Haikara and Enari, 1975). The Schwarz Differential Medium (SDM)
proposed by Brenner et al. (1970) consists of MYGP with 0.3 -0.35% (w/v)
fuchsin-sulphite mixture (4 g basic fuchsin , 25 g anhydrous sodium sulphite and 1
g dextrin) as the only selective agent and permits the growth of the spoilage
organisms S(I&. dfipsoicfeus and &c&c/r. ~firrstrttims, now both recognized as SN&.
rwmisiac (Yarrow, 1984). However, Lin ( 1973) found that several culture yeasts,
representing both ale and lager yeasts, were capable of growth on SDM into
colonies difficult to distillguis~l from the wild yeast colonies.
A combill~tioll between the crystal violet medium and the SDM medium was
suggested by Lin (1974). Lins wild yeast medium (LWYM) contains 0.4 mp crystal
violet and 1.0 g fuchsin-sulphite mixture per litre and was originally recommended
to be used for various species of both Succlluroll?~~(.(,s and no~~-Snc~/?u/.orn~,~c.r wild
yeasts. The level of the crystal violet in the LWYM is much lower than in the
original crystal violet medium and it is recommended that each culture yeast should
be tested on the medium before use in order to lind the optimum concentration of
crystal violet added (between 0.2 and 6 ppm). According to lngledew and Casey
(1982) LWYM is superior to both the crystal violet medium and the SDM for the
detection of S~l~~lrrrroi??i,c.~~~~wild yeasts.
Lysine medium was formulated by Walters and Thiselton (1953) as a synthetic
liquid medium ~oIlt~iliillg L-( + )-lysine as the sole nitrogen source for detection of
Ilon-S~rccltmow??,~~s wild yeasts. The medium was later modified by Morris and
Eddy (1957) as a solid medium. Neither lager and ale yeasts nor most othcl
S~~cchc~~ony~~~~.~ yeasts are capable of iysine utilization and therefore cannot grow on
this medium (Walters and Thiselton, 1953; Seidel, 1972). The yeast culture must bc
washed before inoculating in order to remove extraneous nutrients which may
support the growth of the culture yeast (Walters and Thisclton. 1953). An improve-
ment of the lysinc medium (multi-nitrogen source medium, CLEN, containing
cadaverine, lysinc, ethylamine and nitrate as the sole nitrogen sources) was recorn-
mended by Martin and Siebert (1992). Compared to lysine medium CLEN medium
supports the growth of a larger number of wild yeasts which also grow faster on
CLEN medium than on the lysine medium.
The use of 550 ppm CuSO, added to commonly used yeast media as MYGP, WA
or yeast extract, peptone, dextrose agar (YPD) for detection of non_Suc~cilrn_ot71.~,~~~.s
wild yeasts was introduced by Lin (19X 1). Later Taylor and Marsh (1984) found
that by reduction of the CuSO, concentration to 200 ppm both non-S~ic,c,hurw?lJ:ct.s
and some S~lcc,itrr~onl;t.~~~~wild yeasts could be detected. Bendiak (1991) found that
a CuSO, concentration as low as 50 ppm was sufficient for suppression of culture
yeasts as long as a synthetic medium without amino acids was used instead of
MYGP. The use of MYGP with 200 ppm CuSO, is now recommended by the
American Society of Brewing Chemists (Anonymous. 1992b). The composition is
shown in Table 7.
XMACS medium containing xylose, mannitol, adonitol, cellobiose and sorbitol
as the only carbon sources has been reported to be useful for the detection of some
of both non-Saccharornyces wild yeasts and Succharonyes wild yeasts (de Angelo
and Siebert, 1987). Most brewing yeasts are not able to assimilate the carbon
sources included in this medium, however, some strains will be able to assimilate
one or two, typically mannitol or sorbitol and in this case the medium must be
re-formulated omitting these carbon sources.
The use of other media has been reported including non-selective media contain-
ing actidione at a level of approx. 0.16 ppm (Ingledew and Casey, 1982), non-selec-
tive media with 100 ppm cinnamic acid for specific detection of wild yeasts
producing phenolic off-flavours (Hope, 1987), wort agar adjusted to pH 4.5 and
incubated at 37C for the detection of Sac&. ~j~s~af~~~s, now Sac&. cere~is~~e
(Yarrow, 1984), in lager yeasts (R&ken and Schulte, 1986). Wild yeasts causing
super-attenuation may also be detected on media containing dextrin as the only
carbohydrate (Ingledew and Casey, 1982). As most ale yeasts have an obligate
requirement for panthotel~ate, a panthotenate free medium can be used for the
detection of both Sacchurom~~ct~sand non-S~~~~?~ro~~ces species in ale breweries
(R&ken, 1983). Wallerstein laboratories nutrient (WLN) agar, although originally
proposed for the detection of respiratory deficient yeasts, may also be used for
detection of wild yeasts in ale yeast cultures. The medium inciudes 22 ppm
bromocresol green which can be reduced by various species of both Saccharomyces
and non-Saccharomyccs wild yeasts as well as lager yeasts which then form pale
green, bluish or white colonies. Ale yeasts fail to reduce the bromocresol green and
form dark green colonies (Hall, 1970: Hall, 1971).
Brewing yeast strains other than the actual culture yeast are also regarded as wild
yeasts and Fernhndez et al. (1989) developed a method by which cross-contamina-
tion between lager yeast strains could be detected. The method is based on the fact
that even among closely related lager yeast strains small phenotypic differences can
be detected when using combinations of inhibitory substances. The method includes
various combinations of media such as Lins base agar with 200 and 300 mg CuSO,
per litre respectively, UBA with 200 mg bromocresol green per litre, and dextrose
agar with 10 mg basic fuchsin, 400 mg streptomycin S and 0.03 mg actidione per
litre.

Composition of MYGP with 200 ppm C&O,

Malt extract i&T


Yeast extract 3g
Ghlcose 10 g
Peptone 5g
C&O,-5H,O 312 mg
Agdr 20 &
Distilled water, ad 1000 ml
Final pH 6.2

Taylor and Marsh, 1984; Anonymous, 1992b.


As stated no single medium exists for the detection of wild yeasts and
the combination of media will vary depending on the type of culture yeast
used. For general use a combination of MYGP with 200 ppm CuSO,, a non-
selective medium incubated at 37C and XMACS medium, lysine medium
or CLEN medium should be capable of detecting the majority of both Sacchu-
rom~vx.r and non-Succharon~yws wild yeasts in lager breweries (Rocken and
Schulte, 1986; de Angelo and Siebert, 1987; Martin and Siebert, 1992). All
the media proposed for detection of wild yeasts are based on selective princi-
ples and it is important to bear in mind that the inhibition of the culture yeast
and growth of the wild yeasts will be influenced by the physiological condition
of the yeasts, the cell concentration and incubation conditions (Fernandez et al.,
1989).

4. Conclusioti

In modern breweries the risk of microbial beer spoilage is associated


with growth of specific strains of L~~ctohacil1u.s spp., Pedincoccus spp. as well
as a group of strictly anaerobic Gram negative bacteria which is apparently
gaining increasing significance due to improved beer handling and bottling
techniques which significantly reduce the oxygen content of beer. Another impor-
tant factor is the increased production of beers with low alcohol content.
In addition wild yeasts, in particular S~lc~~hrno,lz~,~~~,~spp. still constitute a signifi-
cant risk.
The ability to grow in beer varies between strains of the same species of
specific spoilage organisms. Experience has shown that very low- levels of infec-
tions may eventually result in growth and spoilage. Further, microorganisms in
beer seem to be specifically adapted to this particular environment and are
sometimes very difficult to grow in other environments including laboratory
media. Microorganisms in beer and brewery samples are often damaged by the
hostile environment and difficult to detect even in enriched media. Apart from
explaining the difficulties involved in establishing effective microbiological quality
control these experiences explain why rapid microbiological methods have not
found their way into the brewing industry.
Breweries today are bound to USC c~~ll~~entional techniques and among the
several media available those most optimal for a particular brewery must be
selected on the basis of a detailed microbiological knowledge and understanding
of the process and the beer produced. However, the quality control should in
most cases at least include a substrate for detection of lactic acid bacteria as e.g.
modified NBB, a medium for detection of strictly anaerobic Gram negative
bacteria as e.g. pre-reduced NBB or SMMP, and a medium for detection of wild
yeasts as e.g. MYGP with 200 ppm CuSO,. Besides, the efficiency of cleaning
and disinfection should be controlled, for this purpose PCA can be used for
determination of a mesophilic aerobic count.
L. Jespersen. M. Jakobsen / Int. J. Food ~i~r~biolo~~ 33 (1996) 139- 155 153

The valuable assistance given by Dr Janet Corry in critically reviewing this


manuscript is highly appreciated.

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