Microbiological Analyses of

Dry and Slurry Yeasts for Brewing

Marisa Manzano 1,2, Cristina Giusto 1, Ingrid Bartolomeoli 1,
Stefano Buiatti 1 and Giuseppe Comi 1

ABSTRACT 4.7. Only a few bacteria are able to grow under such in-
hospitable conditions and are able to spoil the beer 13. To
J. Inst. Brew. 111(2), 203–208, 2005
prevent microbial contamination during the production
Classical microbiological methods in association with molecular process, the microbiological purity of brewing yeast start-
methods (DNA amplification, Temperature Gradient Gel Electro- ers is a necessary condition to maintain high product qual-
phoresis (TGGE) and Denaturing Gradient Gel Electrophoresis
ity. Many strains are available on the market and their
(DGGE) were used. These methods, developed to rapidly ana-
lyze microbial communities on the basis of sequence-specific characterization is necessary for quality control in dry
separation of DNA amplicons, allowed the detection of DNA yeast production 7. The contamination of production strains
differences in the amplicons tested and the identification of the with wild yeasts (non-Saccharomyces or Saccharomyces)
strains analyzed by the comparison of unknown sequences with and bacteria may contribute negatively or positively to
sequences of known species. TGGE allowed the comparison of beer properties and characteristics. Monitoring of micro-
the different Saccharomyces cerevisiae strains used in brewing bial contaminants during the brewing process is important
while DGGE allowed the identification of lactic acid bacteria to obtain reproducible and high-quality beers 16. The plate
(LAB) in beer. These methods are a reliable tool for fast com- count method for enumerating microbiological contamina-
parison of strains of Saccharomyces cerevisiae collected from tion has remained unchanged for over a century, but it
different craft breweries where they were used as starters to
requires several days before the microorganisms are iden-
check the presence of possible yeast contaminants in the brew-
ing process and for rapid LAB identification. tified after plating 17. Consequently, the products are often
already released for sale before the microbiological re-
Key words: Beer, lactic acid bacteria, PCR-DGGE, PCR-TGGE, sults become available. Polymerase Chain Reaction (PCR)
Saccharomyces cerevisiae.
assays have emerged for the specific detection of micro-
bial contaminant in beer and primers for the most com-
INTRODUCTION mon spoilage organisms, especially for lactic acid bacteria
have been designed 6. The PCR technique is a fast and
At present beer is the most common beverage in the more specific method to detect bacteria in beer compared
world after tea, soft drinks and milk 1. Beer production has to classical microbiological methods which are less spe-
become more reproducible and product quality more con- cific and more time consuming. Also real time PCR was
sistent in the last years and the use of yeast starters has used to improve sensitivity in bacteria detection from beer
been essential in beer production since 1883 4. Saccharo- and to date this technique still needs to be applied in rou-
myces cerevisiae is the prevalent yeast species involved in tine brewery practice 15.
brewing and it influences the quality and the quantity of Restriction fragment length polymorphism (RFLP) pat-
the secondary products formed during the alcoholic fer- terns of PCR-amplified DNA fragments and Temperature
mentation. Different yeast strains will produce different Gradient Gel Electrophoresis (TGGE) of PCR products
levels of esters and higher alcohols under similar condi- were successfully used for the identification of yeasts iso-
tions and these compounds will directly influence beer lated from wine 9 and PCR-DGGE was described as useful
flavour. Some yeasts appear to be more vigorous in com- in identifying bacteria during sausage fermentation 3. The
peting for nutrients than others and therefore leave less aim of this work was to identify and quantify the presence
highly assimilable nitrogen and fermentable residue in of wild yeasts and lactic acid bacteria contamination in
beer. Such beer may be less susceptible to microbiological dry and slurry yeasts used in beer production, and to in-
contamination 11. Beer is a poor and rather hostile environ- vestigate intraspecific differentiation of Saccharomyces
ment for most microorganisms: its ethanol concentration cerevisiae strains by PCR-TGGE.
is usually around 4–5% v/v and its pH ranges from 3.8 to

MATERIALS AND METHODS

1 Department of Food Science, via Marangoni 97, 33100 Udine, Italy. Samples of dry and slurry yeasts used in three different
2 Corresponding author. E-mail: marisa.manzano@uniud.it micro-breweries (named A, B and C) in the Friuli Venezia
Giulia region (Italy) were analyzed to evaluate the con-
Publication no. G-2005-0831-285 tamination due to wild yeasts and lactic acid bacteria dur-
© 2005 The Institute of Brewing & Distilling ing the brewing process. Two dry yeast samples, b1 (lager

VOL. 111, NO. 2, 2005 203

yeast) and b2 (same brand, different yeast strain, ale cubated at 30°C for 3 d under microaerophilic conditions.
yeast) obtained from the brewery coded B, were used for After incubation the number of colonies was counted and
one fermentation each, and analyzed before and after in- based on the data the total number of CFU/g was calcu-
oculation in the wort. Two samples of slurry yeasts (a and lated.
c) obtained as “pitching yeast” (lager yeasts) harvested
from the bottom of a fermenter no more than 12 h prior to Choosing yeast colonies for molecular analyses
sampling, and utilized respectively in the breweries coded Yeast colonies grown on both media, WL agar and Ly-
A and C were analyzed before and after their utilization in sine medium, were carefully screened and those which
the fermentation process. Yeast a was used for the follow- were visually morphologically different were selected for
ing eight fermentations (aF1, aF2, aF3, aF4, aF5, aF6, aF7 further analysis. They were purified onto YPD agar me-
and aF8). Yeast c was used for the following three fermen- dium (Oxoid, Milan, Italy) (72 h at 28°C) and pure col-
tations (cF1, cF2, and cF3). Samples from the pitching onies were stored in YPD broth (Oxoid, Milan, Italy)
yeast and samples after the fermentations were subjected supplemented with 30% glycerol (Sigma, Milan, Italy) at
to morphological, biochemical and molecular analyses. –80°C until use. Bacteria were grown overnight at 30°C
on MRS agar (Oxoid). Three bacterial colonies of differ-
Morphological assessment and viability ent morphology were selected from the plate, observed
determination under the microscope and subjected to Gram staining and
One g of each of the samples a and b were transferred catalase activity assay. Gram positive and catalase nega-
to 9 mL of physiological solution (kept at 30°C for 1 h tive colonies were purified onto MRS agar plates and pure
prior to analysis) to obtain a 10–1 preliminary dilution. To colonies were transferred into MRS broth supplemented
revitalize yeasts, the dilution was vortexed 4 times for 30 s with 30% glycerol and stored at –80°C until use.
every 5 min. Dilutions (up to 10–8 ) were prepared. One
drop of the 10–3 dilution was put onto a Bürker camera Galactose fermentation
and observed using a light microscope Axiophot (Zeiss, To differentiate S. cerevisiae strains the fermentation of
Milan, Italy) for morphological analyses. To assess the galactose test was performed. Substrate (7 mL) for fer-
dry yeast viability, a 1 mL vial of the 10–3 dilution was mentation tests (peptone 5%, yeast extract 0.7%) was sup-
thoroughly mixed with a 1 mL vial of Methylene Blue plemented with 2% galactose (Sigma, Milan, Italy) and
staining solution by shaking for 30 sec and again after 10 added to a tube containing a Durham tube. An inoculum
min. The mixture was observed with a Bürker camera of 0.1 mL with each strain isolated for the molecular
using a light microscope (40×)12. Based on the results, the analyses was prepared and tubes were incubated at 28°C
viability of strains was calculated (the percentage of liv- for 15 d. After this time the presence of gas bubbles in the
ing cells compared to their total number). Results were Durham tube indicated that the fermentation of galactose
reported as number of live cells /g dry weight. To stan- occurred.
dardize the analysis for the slurry yeasts, it was necessary
to dry an aliquot of yeast and determine the dry weight DNA extraction from yeast and bacteria
using a thickener balance (Mettler PM 400, Switzerland).
Yeast and bacteria control strains used in this study to
Evaluation of the number of Saccharomyces optimize the TGGE and DGGE protocols are reported in
cerevisiae strains and wild yeasts Table I.
To evaluate the presence of S. cerevisiae strains, 0.1
Table I. Reference strains used.
mL of each dilution starting from 10–5 and higher was
surface-plated on WL agar medium (Oxoid, Milan, Italy). Yeasts Lactic acid bacteria
Plates were incubated at 28°C for 5 d. After incubation the Saccharomyces cerevisiae Lactobacillus sakei DSM° 6333
number of colonies appearing 5 mm in diameter and from ATCC* 36024 Lact. casei DSM° 20011
S. cerevisiae UCDVE $ 812 Lact. curvatus DSM° 20019
cream-coloured to light green 2 was counted and, based on S. cerevisiae UCDVE $ 926 Lact. brevis DSM° 20054
the data, the total number of CFU /g was calculated 18. Cell S. bayanus DSM° 70412 Lact. plantarum DSM° 20174
morphology was analyzed using a light microscope. S. bayanus DSM° 3774 Lact. hilgardii DSM° 20176
To detect and evaluate presence of non-Saccharomyces S. pastorianus DSM° 6580 Lact. paracasei
wild yeast strains, lysine medium (Oxoid, Milan, Italy) S. pastorianus DSM° 6581 Pediococcus pentosaceus
S. paradoxus DBVPG + 6411 DSM° 20336
supplemented with 10% lactic acid (Oxoid, Milan, Italy) Weissella kimkii
was applied following the instructions of the manufac- Kokuria kristinae DSM° 20032
turer. Plates were incubated at 25°C and after 5 days the Kokuria varians DSM° 20033
number of colonies forming units (CFU) was determined. Leuconostoc cremoris DSM° 4196
Lactococcus lactis DSM° 2345
Oenococcus oeni DSM° 20252
Detection and evaluation of the number Enterococcus hirae
of lactic acid bacteria (LAB)
*American Type Culture Collection, Manassas, Virginia, USA.
To detect and evaluate the presence of LAB, 1 mL of $ University of California Department of Viticolture and Enology, Davis,

dilutions from 10–1 to 10–4 were transferred to duplicate USA.

°Deutsche Sammlung von Mikrorganismen und Zellkulturen, GmbH,
plates of MRS agar (Oxoid, Milan, Italy) supplemented Braunschweig, Germany.
with the antifungal/antimould agent Delvocid ® Instant § Department of Microbiology, Zagreb, Croatia.
(DSM Food Specialties, The Netherlands). Plates were in- + Dipartimento di Biologia Vegetale, Perugia, Italia.

204 JOURNAL OF THE INSTITUTE OF BREWING

 Yeast pure colonies were cultured overnight at 30°C in tham, Massachusetts, USA) in a final volume of 50 µL
YPD medium and centrifuged at 6000 rpm for 10 min at using the primers P1 5⬘-CGCGCGTGCCTAATACA-
10°C. The pellet was washed with sterile, cold, deionised TGC-3⬘ with a GC clamp and primer P2 5⬘-TTCCCC-
water and subjected to the DNA extraction as described ACGGCTTACTCACC-3⬘ which targeted the V1 region
by Manzano et al.9 of the 16S rRNA according to the protocol proposed by
Bacteria pure colonies were cultured overnight in MRS Cocolin et al.3 PCR fragments were analyzed by electro-
broth and centrifuged at 6000 rpm for 10 min at 10°C. phoresis in a 2% agarose gel as reported above.
The pellet was washed with sterile deionised water and
subjected to DNA extraction as described by Manzano et DGGE analysis of bacteria
al.8 To eliminate presence of RNAs, RNase (Sigma, Mi- The Dcode universal mutation detection System (Bio-
lan, Italy) was added to the DNA (incubation for 1 h at Rad Laboratories, Richmond, USA) was used for a
37°C). Concentration of DNAs extracted from yeasts and DGGE analysis of the PCR products obtained from single
bacteria was measured using a SmartSpec TM 3000 spec- colonies. Electrophoresis was performed in a 0.8-mm
trophotometer (Bio-Rad, Milan, Italy) and standardized at polyacrylamide gel (8% (w/v) acrylamide-bisacrylamide
~50 ng µL–1 before being used in the PCR assay. Samples (37.5:1)) using the denaturant gradient from 40% to 60%
were stored at –20°C prior to subsequent analyses. increasing in the direction of the electrophoresis. The gels
were subjected to a constant voltage of 130 V for 3.5 h at
PCR amplification for a subsequent TGGE 60°C, and after electrophoresis they were stained as re-
analysis of yeast isolates ported for TGGE.
Primers Shaf + GC and Shar designed to amplify a
fragment of 207 bp between Internal Transcribed Spacers RESULTS AND DISCUSSION
(ITS) 1 and 2 were used: forward primer Schaf (5⬘-GTA-
GTGAGTGATACTCTT-3⬘) and reverse primer Schar Classical microbiological methods
(5⬘-AGAACATGTTGCCTAGAC-3⬘) were designed and The dry yeasts b1 and b2 showed a CFU /g of 2.8 ×
annealing sites and expected PCR amplicon sizes were 1010 and 5.5 × 109 respectively while the values obtained
checked with the “Amplify” (Madison, Wisconsin, USA) for samples a and c analyzed before being inoculated in
program. Schaf primer anneals from 18 to 38 bp and Schar the wort were respectively 5.0 × 107 and 5.3 × 109 CFU /g.
primer from 210 to 229 bp giving a fragment length of The viabilities of the b1 and b2 dry yeasts were 67% and
211 bp. In order to use the resulting amplicons for TGGE 73% respectively. No non-Saccharomyces wild yeasts that
a “GC-clamp” (5⬘-CGCCCGCCGCGCGCGCGGCGG- could grow on lysine media were present in the starters as
GCGGGGCGGGGGCACCGCGCG-3⬘) was added to indicated by the absence of growth.
the 5⬘ end of the Schaf primer 9. On the basis of the appearance of the typical S. cere-
PCR amplification was carried out in a 50 µL reaction visiae morphological characteristics, 44 S. cerevisiae
mixture according to the protocol proposed by Manzano strains were picked up, purified and tested. Of the 44 iso-
et al.9. A 5 µL aliquot of the product was separated elec- lated and tested S. cerevisiae strains, 38 were able to fer-
trophoretically in a 2% agarose gel (Sigma, Milan, Italy) ment galactose.
stained with ethidium bromide (0.5 µg mL–1 ) in 0.5× TBE In spite of the same LAB contamination level shown
buffer (0.045 M Tris borate, 0.001 M EDTA pH 8) and by two dry yeast starters (b1 and b2) before their utiliza-
compared with Molecular Weight Marker 100 bp (Sigma,
Milan, Italy).
TGGE analysis of yeasts
Yeast strains reported in Table I were used to optimize
the PCR-TGGE protocol used to differentiate different
strains obtained from beer fermentation processes.
The DCode Universal Mutation System (Bio-Rad Lab-
oratories, Richmond, USA) was used for the sequence
specific separation of the PCR products. Electrophoresis
was performed in a 0.8-mm polyacrylamide gel (8% w/v)
acrylamide-bisacrylamide (37:5:1) (Fluka, Germany) con-
taining 7 mol L–1 urea (Fluka). The gels were subjected to
a constant voltage of 120 V for 4.5 h from 56.5 to 60.5°C
with 0.8°C/h temperature ramp for yeasts. After electro-
phoresis gels were stained for 20 min in a 1.25× TAE
(0.04 M Tris acetate, 0.001 M EDTA pH 8) containing 1×
(final concentration) SYBR Green (Sigma, Milan, Italy),
visualized under UV light and filed using the GeneSnap
Syngene Software (Cambridge, UK). Fig. 1. TGGE of colonies isolated from brewery B on WLN
PCR conditions for bacteria media. Lane 1: b1F1.2; lane 2: b1F1.1; lane 3: b2F1.3; lane 4:
b2F1. 2; lane 5: b2F1.1; lane 6: b2.3; lane 7: b2.2; lane 8: b2.1;
Amplification was performed with a Peltier Thermal lane 9: b1.2; lane 10: b1.1; lane 12: Saccharomyces cerevisiae
Cycler, PTC 220 DNA engine Dyad (MJ Research, Wal- ATCC 36024.

VOL. 111, NO. 2, 2005 205

 A B
Fig. 2. A. TGGE samples isolated from brewery A; lane 1: Saccharomyces cerevisiae ATCC 36024; lane 3: Saccharo-
myces cerevisiae 926; lane 4: Saccharomyces cerevisiae 812; lane 5: a.1; lane 6: a.2; lane 7: a.3; lane 8: aF1.1; lane 9:
aF1.2; lane 10: aF2.1; lane 11: aF2. 2; lane 12: aF3.1; lane 13: aF3. 2; lane 14: aF4.1; lane 15: aF4.2; lane 16: aF4.3.
B. TGGE samples isolated from brewery C; lane 1: Saccharomyces cerevisiae ATCC 36024; lane 2: c.1; lane 3: c.2;
lane 4: c.3; lane 5: c.4; lane 6: yeast cF1.1; lane 7: cF1.2; lane 8: cF2.1; lane 9: cF2.2; lane 10: cF2.3; lane 11: cF3.1;
lane 12: yeast cF3.2.

Fig. 3. Standard LAB DGGE; primer P1/P2. Lane 1: Lb. plantarum DSM 20174; Lane 2: Lb. sakei DSM 6333; Lane 3:
Lb. curvatus DSM 20019; l Lane 4: Lb. brevis DSM 20054; Lane 5: Lb. casei DSM 20011; Lane 6: Lb. paracasei; Lane
7: Pediococcus parvulus DSM; Lane 8: Pediococcus pentosaceus DSM; Lane 9: Enterococcus hiirae; Lane 10: Oeno-
coccus oeni; Lane 11: Lb. hilgardii DSM 20176; Lane 12: Lc. lactis DSM 2345; Lanes 13–14: Kokuria kristinae DSM
20032; Lane 15: Kokuria varians DSM 20033; Lane 16: Leuconostoc cremoris DSM 4196; Lane 17: Weissella kimkii.

206 JOURNAL OF THE INSTITUTE OF BREWING

tion (9 × 10 CFU /g), different levels were observed after strains used as starters in beer production. The TGGE
the fermentations. The b1 reached 1 × 102 CFU /g while technique (Figure 3) applied in this work allowed the
b2 reached 2.7 × 106 CFU /g. Different hygienic practices intraspecific differentiation among the strains tested and it
applied to the brewery process influenced the bacterial was possible to differentiate starters on the basis of the
charge of the wort. Brewery C showed the higher LAB size of their amplicons and to detect a “wild” S. cerevisiae
levels detected during the analyses, from 1.7–1.8 × 107 strains not belonging to any of the two starters used in the
(samples cF1 and cF2) to 3 × 107 CFU /g (sample cF3). In brewery B. This culture-independent method allows sepa-
brewery A the values of the LAB were under 1 × 102 ration of DNA molecules that differ by single bases, char-
CFU /g for the slurry yeast before being used in the fer- acterizing analysed strains in a short time. Identification
mentation process and from 1.3 × 102 to 3.3 × 102 after results are available within 8 h after cell growth onto the
the four fermentations (aF1, aF2, aF3 and aF4) while the isolation media used. Moreover the method is useful in
values increased from 3.5 × 104 to the maximum level the direct detection of microbes contaminating foods or
achieved by the sample aF7, 5.1 × 106, during the last beverages without the need of plating or, as reported by
fermentations, from aF5 to aF8. Manzano et al.10 in the monitoring of industrial or craft
fermentation processes. The PCR-TGGE or PCR-DGGE
Molecular methods methods can be used in identification of contaminants or
The single DNA fragments obtained by PCR for two in strain differentiation in a short time without employing
colonies morphologically different (b1.1 and b1.2) iso- complicated biochemical tests.
lated from WL agar plating in the yeast b1 (brewery B), A total of 44 Gram-positive, catalase negative rod or
migrated to the same position in TGGE (Figure 1 lane 10 cocccus-shaped strains isolated from MRS with the addi-
and 9) suggesting the same sequence for both PCR prod- tion of Delvocid were identified by PCR-DGGE (Table
ucts. The amplification products obtained by PCR for II). On the basis of different positions reached by the PCR
three colonies (b2.1, b2.2, b2.3) isolated from WL agar on products in the DGGE the bacteria were identified as
the basis of the different morphologies exhibited by the Lactobacillus brevis, the unique species in the brewery A
yeast b2 showed the same profile in TGGE (Figure 1 lanes and the main species detected in breweries (70%), in ac-
6, 7 and 8). The b1 amplicons (Figure 1 lanes 10 and 9) cordance with the data reported by Juvonen and Satokari 6
migrated to different positions in the gel in respect with and Satokari et al.14 Kokuria, Lactobacillus brevis, Lb. sa-
the b2 amplicons (Figure 1 lanes 8, 7 and 6) allowing us kei and Lactococcus lactis were detected in the brewery B.
to differentiate the two yeasts employed as starters in beer Lactobacillus brevis, Lb. hilgardii and Pediococcus par-
production. After the fermentation (F1) with the starter vulus were recovered in the brewery C 5. No Gram nega-
b1, two colonies (b1F1.1 and b1F1.2) isolated from WL tive catalase negative strains were isolated from aF1 and
agar were subjected to PCR-TGGE analyses. The ampli- aF2 samples demonstrating only lactic acid bacteria were
con obtained, b1F1.2 (Figure 1 lane 1), migrated to the detected in beer samples using microaerophilic sampling
same level of the amplicon obtained by the starter (Figure conditions.
1 lanes 9 and 10), on the contrary the fragment b1F1.1 Most hazardous for the brewing industry are the LAB
(Figure 1 lane 2) migrated to a different level indicating belonging to the genera Lactobacillus and Pediococcus,
that the two colonies isolated after fermentation (b1F1.2 because they can produce haze or rope and cause unpleas-
and b1F1.1) belong to different strains. In the case of the
starter b2, the colonies isolated from WL agar (b2F1.1,
b2F1.2, b2F1.3), obtained by plating the sample after the Table II. DGGE identification of LAB strains isolated before and after
brewing.
fermentation process, showed different TGGE profiles
(Figure 1 lanes 5, 4 and lane 3). Two PCR products ob- Brewery Samples DGGE identification /total isolated
tained from the samples b1F1.1 (yeast b1) and b2F1.3 A* a 3 Lactobacillus brevis /3
(yeast b2) migrated to the same levels demonstrating the aAF 3 3 Lactobacillus brevis /3
same sequence. Since they were isolated from two differ- aF 4 3 Lactobacillus brevis /3
ent fermentation processes it is reasonable to assume that aF 5 3 Lactobacillus brevis /3
the same yeast was present during the two fermentations, aF 6 3 Lactobacillus brevis /3
probably because it was present on the raw material used
aF 7 3 Lactobacillus brevis /3
for the brewing or from the environment and not from the
aF 8 3 Lactobacillus brevis /3
starters used.
All strains isolated from brewery A belong to the same B B1 3 Kokuria kristinae /3
strain of S. cerevisiae used as starter; the TGGE profiles b1F1 3 Lactobacillus brevis /3
are identical for all samples (Figure 2A). Similar results b2 1 Lactococcus lactis /3
were obtained for brewery C (Figure 2B). No correlation 2 Lactobacillus sakei /3
was found between galactose fermentation results and b2F1 3 Lactobacillus brevis /3
strain TGGE differentiation, in fact DNA fragments C c 2 Lactobacillus hilgardii /2*
showed migration profiles typical of S. cerevisiae behav- cF 1 3 Lactobacillus brevis /3
iours independent from the negative galactose fermenta- cF 2 2 Pediococcus parvulus /3
tion obtained. 1 Lactobacillus brevis /3
The use of specific primers for the Saccharomyces cF3 3 Lactobacillus brevis /3
sensu stricto group is a reliable and fast system to identify *From samples aF1 and aF2 no LAB were isolated; from sample c only
and to compare the different Saccharomyces cerevisiae 2 LAB strains were isolated

VOL. 111, NO. 2, 2005 207

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Various contamination levels were present among the DGGE differentiation of Saccharomyces sensu stricto strains.
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10. Manzano, M., Cocolin, L., Iacumin, L., Cantoni, C., Comi, G.,
A PCR-TGGE (Temperature Gradient Gel Electrophoresis) tech-
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208 JOURNAL OF THE INSTITUTE OF BREWING