BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Quality of Life and Management of Living Resources
Development and demonstration of polymerase chain reaction based methods for
process control in brewing industry
BREWPROC
Key action No 1: Food, nutrition and health
1.1 Development of safe and flexible and new and/or improved manufacturing processes and
technologies
1.1.1
1.1.2
1.1.3
Novel and improved biological raw materials for high quality food
Advanced and optimised food technologies, packaging systems and process control
Quality monitoring and traceability throughout the food chain
1
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Table of contents
Section Contents
Page
1.
OBJECTIVES AND EXPECTED ACHIEVEMENTS
3
2.
PROJECT WORKPLAN
4
2.1
2.2
2.3
Introduction
Project structure, planning and timetable
Workpackage descriptions
4
8
14
3.
ROLE OF PARTICIPANTS
20
4.
PROJECT MANAGEMENT AND CO-ORDINATION
28
5.
EXPLOITATION AND DISSEMINATION ACTIVITIES
30
6.
ETHICAL ASPECTS AND SAFETY PROVISIONS
32
ANNEXES
Annex 1.
Time table of project activities by task.
2
BREWPROC
1.
Technical annex
Project proposal Nr QLRT-1999-31251
OBJECTIVES AND EXPECTED ACHIEVEMENTS
The proposed project is a Combined R & D - Demonstration project (Fig. 1). It is aiming to provide
rapid, specific and thoroughly tested PCR based methods for the microbiological monitoring of the
brewing process to allow proactive process control and successful implementation of the PCR
technology in the brewing industry in Europe. The project will bring together research scientists with
experience in applying PCR technology in the area of brewing microbiology and a SME operating in
the field of PCR kit development with breweries which belong to the opinion leaders in the brewing
industry in Europe.
Over-all objective of the project is to improve microbiological quality and safety of beer produced in
Europe through implementation of polymerase chain reaction (PCR) technology in brewery quality
control.
Brewing process
BEER
Dissemination of knowledge
filtration
Demonstration phase
Performance and applicability to
routine quality control
FERMENTING
WORT
PCR methodology development and
improvement
PITCHING YEAST
R & D phase
Time
Figure 1. Development and demonstration of polymerase chain reaction based methods for process control in breweries the project outline.
R & D objectives
R & D phase aims to improve microbiological monitoring of brewery samples with PCR based
methods. It is expected that the PCR based methods established in this project will allow faster and
more specific detection of the contaminants along the production line than those at present in use in
breweries. Wrong alarms caused by non-spoilage organisms will be minimised since only spoilage taxa
are revealed. PCR will also facilitate the detection of fastidious organisms.
Specific objectives are:
 To improve PCR detection of spoilage organisms in beer. Comparative data on the technical quality of
the existing PCR assays will be provided to facilitate the standardisation and optimisation of the methodology. The
sensitivity of the assays will be improved by enrichment and PCR optimisation to allow detection of presence of
spoilage organisms in beer. The reliability will be improved internal/external standardisation of the PCRs. New primers
will be provided to enable detection of all spoilage taxa of importance in a most effective way. The advances in the
methodology development will be exploited for the formulation of PCR kit prototypes. As a result, PCR based assays
possessing an adequate level of sensitivity, simplicity and specificity to be introduced to brewery QC laboratories will
ensue.

To develop novel PCR based methods for the detection of spoilage organisms in unfiltered
process samples. It is expected to provide novel nucleic acid amplification based methods for microbiological
monitoring of unfiltered process samples for the presence of various spoilage organisms. New oligonucleotides will be
designed for reliable recognition of specific process contaminating organisms and simple and effective pre-PCR sample
treatment methods will be created. Methodology development will lead to a formulation of PCR kit prototypes for
process samples. It will be also determined if specific RNAs can be employed as a determinant of viability of brewery
contaminants.
3
BREWPROC

Technical annex
Project proposal Nr QLRT-1999-31251
To improve and facilitate the analysis of brewery contaminant specific PCR amplicons. This
project is expected to provide PCR product analysis methods that are automated/amenable to automation, allow
confirmation, unequivocal interpretation and quantification of the result, high sample throughput and are safe to use.
Different detection methods and assay formats based on fluorogenic on-line detection and microplate hybridisation will
be established and compared to each others.
Demonstration objectives
Demonstration phase aims to enable the wide acceptance of the PCR technology and its successful
implementation in the microbiological quality control in the European brewing industry.
Specific objectives are:
 To establish the validity of the PCR technology for microbiological monitoring of brewery
samples. The technical performance of the developed PCR tests and kit prototypes will be evaluated through
independent testing in research and brewery QC laboratories in comparison with the established methods. In the method
comparison, spiked as well as real production samples will be used, since a lot of negative test results would be
expected. Through comparison of the results from both methods, a reliable estimate of the technical quality of the new
PCR tests will be achieved. Considerations will be relative accuracy, specificity and limit of detection.

To demonstrate the applicability of the PCR technology to brewery quality control. The main
emphasis will lie on the demonstration of the practicability of the PCR technology and its novel applications in brewery
QC laboratories where the local, technical and personnel circumstances differ from those in a research laboratory. The
industry evaluation will provide valuable information about the applicability of the new PCR tests to brewery QC with
regard to their user-friendliness, sampling capacity, sensitivity to DNA contaminations, ease of adoption, suitability to
laboratory routines and safe usage. Besides, the general acceptance of a new DNA based technique in the field of
microbiological QC of food will be improved. Breweries will get also practical experience about PCR work.

To prove the benefits of the PCR technology in brewery quality control. For the time being, the
possible benefits following from the application of PCR can only be estimated, since no data on real advantages of
PCR over the established methods in routine QC is available. In this project, the breweries will evaluate the benefits of
PCR both in the analysis results and in costs. The demonstration of real benefits will facilitate the transfer of the new
technology to the end-users. The results can be also used as a basis for further application of PCR in other fields of
food production.

To create an early awareness of the possibilities of PCR in routine food control. To facilitate the
adoption and acceptance of the PCR technology results will be disseminated to authorities, future end-users and other
potential exploiters Europe-wide in meetings and symposia targeted at different groups, in World Wide Web and in
relevant technical and scientific journals, as appropriate. The existing network of the European Brewery Convention
(EBC) will act as a key dissemination channel. Training workshops will be an important means of distributing the
theoretical and practical skills needed for implementation of the PCR technology into industrial QC laboratories. In
addition, the established methods will be incorporated into the EBC Analytica Microbiologica, the Institute of Brewing
(IOB) Methods of Analysis and MEBAK (Mitteleuropäische Brautechnische Analysenkommision).
2.
PROJECT WORKPLAN
2.1
Introduction
The proposed project is a combined R & D - Demonstration project. The workplan has been broken
down into a R & D and a Demonstration phase both consisting of three interactive workpackages
(WPs) (Fig. 2, page 13). The R & D phase concentrates on the development and establishment of PCR
based methods and kit prototypes which are sensitive, user-friendly and robust enough to be used in
brewery routine QC. Development work will cover the different sample types analysed in the brewing
process. The viability of the resulting methods and the real benefits following from the use of PCR
technology in QC control in breweries will be thoroughly evaluated in the Demonstration phase. The
results from the project will be disseminated to all potential future exploiters covering the whole food
industry and to other interest groups.
4
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
R& D phase
WP1 (PCR based detection methods for spoilage organisms in bright beer) is directed at establishing PCR
based methodology for the microbiological QC of bright beer. P3 will compare the simple PCR
methods existing for detection of Lactobacillus, Megasphaera and Pectinatus bacteria in beer in terms
of their limit of detection, specificity, ease of use and reliability using pure cultures and spiked and
non-spiked beers provided by P10. Based on the results of the method comparison, the consortium will
select the best practice procedures for the PCR detection of the above mentioned bacteria. P1, P2 and
P4 will work on internal standardisation of the specific PCRs. Subsequently, P1 and P2 will test and
optimise various physical and growth based enrichment procedures to improve the sensitivity of the
PCR detection systems to < 10 cells per 500 ml beer. P1, P2 and P4 will design and validate internally
standardised PCRs for those beer spoilage taxa for which PCR tests are not yet available. The pre-PCR
treatment steps will be adjusted, if necessary, for analysing the new target organism taxa in beer and
evaluated. P4 will directly utilise the results from the sample preparation methods and the primer sets
for the development of kit prototypes for PCR detection of spoilage organisms in bright beer. The
viability of the developed methods and kit prototypes will be demonstrated in the Demonstration part.
The primers and the sample treatment methods will be provided for use in WP2 and WP3.
WP2 (PCR based detection methods for spoilage organisms in unfiltered process samples) will focus on the
development of PCR based assays for spoilage organisms in unfiltered process samples. Specific
primers will be designed for primary contaminants with the incorporation of internal standards (P1, P2,
P4). The target organisms will include, for example, pediococci and wild yeasts. The work will focus
on the development of simple, sensitive and rapid methods for the preparation of DNA from process
samples. For DNA isolation simple modifications of conventional procedures and commercial kits will
be tested beside the methods from WP 1. Three approaches will be applied to optimise the sensitivity:
1) the use of separation and concentration techniques, 2) nested PCR, and 3) pre-enrichment. P4 will
use the PCR methods and primers to produce PCR kit prototypes useable in QC laboratories to detect
spoilage organisms in unfiltered samples. The developed PCR methods and kit prototypes will be
provided for use in WP1 and WP3 and evaluated in the Demonstration phase of the project.
P3 together with P1 will study the suitability of reverse transcriptase (RT)-PCR for the selective
detection of viable brewery contaminants by targeting rRNA and translation elongation factor mRNAs.
The cells will be subjected to different killing treatments, and analysed by RT-PCR and Northern
hybridisation after various post-treatment times. In comparison, the persistence of DNA in similarly
treated cells will be studied by DNA-PCR. The effect of sample matrix and cell´s physiological state on
the disappearance of RNA and on the sensitivity of the RT-PCR will be determined, respectively. If
preliminary experiments prove successful, P3 will develop a procedure for the detection of brewery
contaminants in process samples.
WP 3 (Development and application of improved detection methods for PCR amplicons) is directed at the
development of PCR product analysis methods which are better suited to routine diagnostics than
electrophoretic methods. P2, P4 and P8 will set up 5´-nuclease and Hyb-probe assays for the
fluorogenic on-line detection of PCR amplicons. Primers and probes will be designed for various
spoilage bacteria and yeasts, if those developed in WP1 and 2 will be found incompatible with the online systems. The PCR conditions will be optimised for the on line assays. Thermocycling and the
analysis of PCR products will be carried out using a LightCycler (Roche) or TaqMan system (PE
Applied Biosystems).The specificity and sensitivity of the assays will be elucidated using pure cultures
of target and non-target organisms and spiked brewery samples. The previously developed sample
treatment methods will be used when possible. The applicability of the assays to the analysis of real
production samples will be determined by P7 and P8. The results of the on-line assays will be
compared with electrophoretic methods and/or ATP-bioluminescence. P3 will develop microtitre plate
hybridisation for the post-PCR analysis. Internal oligonucleotide probes for the identification of the
PCR amplicons specific to various brewery contaminants will be designed. The capture oligonucleotide
will be covalently linked to the microtitre plate. The detection oligonucleotide will be labelled with
biotin. The hybrid molecules will be detected by avidin conjugated with enzyme and a chromogenic
5
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
substrate. The colour reaction will be measured in a microtitre plate reader. The combination of
sandwich hybridisation and PCR (PCR-ELISA) will be evaluated in terms of sensitivity and specificity
in comparison with Southern hybridisation and electrophoresis.
The developed assays will be compared in terms of their technical and practical attributes by analysing
a set of spiked samples in parallel with all methods (P3, P4, P7, P8). The number of methods evaluated
depends on the technical advances achieved. A report about the comparative study will be proceeded
for dissemination in WP6.
Demonstration phase
WP4 (Method comparison study). The key objective is to independently assess the intrinsic technical
qualities of the PCR based assays and kit prototypes developed during the R & D phase (WP1 and
WP2) for the detection of spoilage organisms in bright beer and in process samples. Detailed objective
analyses procedures will be designed by a statistician (SC1) in conjunction with P1 and P10. Brewery
contaminants will be collected by SC10 from other partners for the preparation of the reference
material and will include e.g. acetic and lactic acid bacteria, Megasphaera, Pectinatus and wild yeasts.
This set of test organisms will be added to an ECCO (European Culture Collections´ Organization)
culture collection to be held by SC10 for future use by brewing microbiologists. The samples will be
defined in terms of microbial composition, inoculum level and viability as directed in the experimental
plan. Samples will be posted directly to P10 for the method comparison. P10 will verify the proposed
study protocol in a preliminary trial. In the method comparison study, each reference sample supplied
will be analysed by the PCR method under consideration and by the standard methodology (EBC
Analytica Microbiologica, MEBAK). The attributes considered will include limit of detection,
specificity, relative accuracy and ease of use in a specialised laboratory. The data collated by P10 will
be e-mailed to a statistical expert (SC1) for analysis. Progress and final reports will be prepared in
collaboration with the expert laboratory (P10). Reports will be passed to WP5 for reviewing and to
WP6 for dissemination.
WP5 (Industry evaluation) is directed at demonstrating the practicability and the technical performance
of the developed PCR based methods and kit prototypes under QC laboratory conditions, and to show
their real benefits in comparison with routine methods. The study will be chiefly planned and organised
by the research partners. An experimental plan, questionnaires, a training programme for brewery
technicians and detailed instructions for the experiments in the breweries will be provided. The brewery
QC laboratories (P5-9) will be supplied with information about the required laboratory arrangements
and equipmentation, and subsequently will be adapted to PCR work (P1-4). After completion of the
training period, the PCR methods will be established as a part of their routine QC. The brewery
samples to be examined will be divided into two parts and analysed in parallel with the PCR methods
and routine methods. P10 will regularly provide the breweries with positive control samples. The
testing will last over two periods of up to six months each. Data on the practicability and benefits of
the new methodology will be collected through interviews and questionnaires. All work will be
overseen locally by brewery QA management and monitored by the research partners. P1-4 and 10 will
analyse the results in collaboration with SC1 who is responsible of the statistical assessment. Reports
will be provided after defined intervals and passed to WP6 for dissemination.
WP6 (Dissemination of knowledge). The purpose of this WP is to bring about full awareness of the outputs
of the project and to maximise the degree of implementation and exploitation by the relevant producer
and consumer groups across the Community. In the dissemination of the results, resources available
under the organisational framework of the European Brewery Convention (EBC) and appropriate
institutes is used. Several of the partners are active members of the EBC Analysis Committee
Microbiology Subcommittee (P1, P4, P5, P6), the EBC Brewing Science Group (P1, P2, P3, P5, P6)
and the EBC Detection of Contaminants Subgroup (P1, P3, P4, P5). Additionally, the biennial EBC
Congress will be used as a platform for dissemination. This will directly enable efficient
communication concerning the findings of the project within the European brewery industry both
during the ongoing project and after its end.
6
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Scientific publications will be reviewed and a list of references arising from the project will be
prepared and distributed by P5 and P10. Standard methods based on results from WP4 and WP5 will be
prepared and incorporated to the manuals EBC Analytica Microbiologica (P4, P5), IOB Methods of
Analysis (P3, P10) and MEBAK (P2). Web pages for inclusion or linkage to the web sites of brewing
companies, research institutes, the EU and the EBC will be prepared under the responsibility of P5
providing on-line information including contacts and references of publications. Two training
workshops for end-users will be organised during the project by P2, P4 and P10. Workshops are also
arranged after the end of the project according to arising requests. After the end of the project, a special
Symposium on the PCR technology and its applications in the beverage producing industries is
arranged by P1. Representatives from the wine, cider and other beverage industry are invited in
addition to participants from the brewing industry, and the authorities are addressed as well. The
presentations of the Symposium are later published as a monograph.
7
BREWPROC
2.2
Technical annex
Project proposal Nr QLRT-1999-31251
Project structure, planning and timetable
List of participants
Partner *
Nr
P1
SC1
Name
Address
Telephone Nr
Telefax Nr
e-mail
VTT Biotechnology
(please note the new name of the
institute)
VTT Internal Services
P.O. Box 1500,
FIN-02044 VTT, Finland
+358 9 456 5130
+358 9 455 2103
auli.haikara@vtt.fi
+358 9 456 6727
+358 9 456 7015
leila.peltoniemi-laajanen@vtt.fi
+49 8161 713 329
+49 8161 713 101
e.geiger@lrz.tu-muenchen.de
P2
TU München-Weihenstephan,
Lehrstuhl für Technologie der
Brauerei II
P.O. Box 2101
FIN-02044 VTT, Finland
Alte Akademie 3,
D-85350 Freising-Weihenstephan,
Germany
P3
BRi (Brewing Research
International)
Lyttel Hall, Surrey, RH1 4HY
United Kingdom
+44 1737 822 272
+44 1737 822 747
sam.walker@brewingresearch.co.uk
P4
PIKA Weihenstephan GmbH
(please note the new name of the
company)
Scottish Courage Ltd., Technical
Centre
Alte Akademie 3,
D-85350 Freising, Germany
+49 8161 496 5800
+49 8161 496 5801
vogeser@lrz.tum.de
Sugarhouse Close,
160 Canongate, Edinburgh EH8
8DD, Great Britain
Thv. Meyersgt. 9-11
P.O. Box 7152 Maj, 0307 Oslo,
Norway
An der Streue, D-59872 MeschedeGrevenstein, Germany
P.O. Box 107307, Am Deich 18/19,
D-28199, Germany
Dobenanstr. 83, D-08523 Plauen,
Germany
Gipsy Lane Campus Headington,
Oxford OX3 0BP, Great Britain
+44 131 2481 140
+44 131 2481 101
jeff.hodgson@scbrew.co.uk
+47 22 06 9435
+47 22 06 9902
tore.hage@prippsringnes.com
+49 2934 959 680
+49 2934 959 481
stefan.loch-ahring@veltins.de
+49 421 5094 4304
+49 421 5094 4437
slustig@becks.de
+49 1374 1211 103
+ 49 1374 11211 591
thomas.sommer@sternquell.de
+44 1865 483248
+44 1865 483242
kasmart@brookes.ac.uk
23 St. Machar Drive, Aberdeen,
AB24 3RY, Great Britain
+44 1224 273 332
+44 1224 272 461
t.dando@ncimb.co.uk
P5
P6
Ringnes a.s.
P7
Brauerei Veltins
P8
Brauerei Beck & Co,
Qualitätswesen
Sternquell Brauerei Plauen GmbH
P9
P10
Oxford Brookes University, School
of Molecular and Biological
Sciences
SC10
National Collection of Industrial
and Marine Bacteria
* P = Partner, SC = Subcontractor
8
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Table 1. Workpackage list (R & D and Demonstration phase).
Workpackage
no
Workpackage title
Phase
Responsible
participants
no1
Personmonths
Start
month
End Deliverable(s)
month
no
1
PCR based detection methods for
spoilage organisms in bright beer
R&D
1, 2-4, 10
52
1
20
1, 2 , 4-7, 22, 24
2
PCR based detection methods for
spoilage organisms in unfiltered
process samples
R&D
3, 1, 2, 4
73
1
24
3, 10, 11, 27, 29
3
Development and application of
improved detection methods for
PCR products
R&D
4, 2, 3, 7, 8
77
1
34
17, 18, 28, 30,
31, 33
4
Method comparison study
Demonstration
10, 1
30
10
30
13, 14, 16, 23, 32
5
Evaluation in industry
Demonstration
2, 1, 3-10
88
8
34
8, 9, 25, 26, 34
6
Dissemination of knowledge
Demonstration
5, 1-4, 10
41
1
42
15, 19-21, 35, 37
361
TOTAL
1
Workpackage leader listed first
Table 2. List of milestones (R & D and Demonstration phase).
MileTitle
stone
no
R1 PCR tests for Lactobacillus,
Megasphaera and Pectinatus spp.
Estimated
delivery
month
10
R2 RT-PCR evaluation
12
D3 On line information
12
R4 New PCR tests for contaminants in
beer samples
R5 New PCR tests for contaminants in
unfiltered samples
R6 RT-PCR assays for viable spoilage
organisms
D7 Intrinsic technical quality of the
PCR tests for beer
20
R8 PCR-ELISA and on-line methods
34
D9 Industry evaluation results
34
D10 Standard methods
36
D11 PCR symposium
42
R=R & D phase
D=demonstration phase
21
24
30
Partners
1-4
Description
PCR based assays and kit prototypes for testing beer
for the presence of Lactobacillus, Megasphaera and
Pectinatus spp. established.
1, 3 Suitability of RT-PCR detection of rRNA and mRNA
for viability assessment evaluated.
5
On line information of the technology including list of
publications.
1, 2, 4 PCR based assays and kit prototypes for testing beer
for the presence of other spoilage taxa established.
1, 2, 4 PCR based assays and kit prototypes for spoilage
organisms in unfiltered process samples developed.
3
RT-PCR based assays for viable spoilage organisms in
unfiltered process samples developed.
10, SC1, Intrinsic technical quality of the PCR methods and kit
SC10, 1 prototypes in the analysis of spiked beer and process
samples objectively demonstrated.
2-4, 7, 8 Automated on-line systems and PCR-ELISA assays
developed and implemented.
1-10, SC1 Adaptability of the PCR technology to brewery QC
laboratories, the viability of the developed PCR
methods and real advantages of PCR in QC
demonstrated
2-5, 10 Publication of standard method(s) in EBC Analytica
Microbiologica, IOB Methods of Analysis and
MEBAK.
1
Special Symposium devoted to the application of PCR
technology for the brewing industry and extended
audience.
SC=subcontractor
9
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Table 3. List of deliverables (R & D and Demonstration phase).
DeliveDeliverable title
Evaluated Partner
rable
delivery
no1
month
R1 Comparable data on the performance of the simple PCR
4
3
based assays existing for beer spoilage bacteria. Dissemination to WP2, WP3 and WP6.
R2 Provision of internal standards for PCR reactions specific
6
1, 2, 4
to Lactobacillus, Megasphaera and Pectinatus spp..
Dissemination to WP2 and WP3.
R3 Provision of internally standardised PCR reactions
6, 10
1, 2, 4
specific
to
process
contaminating
organisms.
Dissemination to WP1 and WP3.
R4 Provision of internally standardised PCR reactions
9
1, 2, 4
specific to other beer spoilage taxa. Dissemination to
WP2 and WP3.
R5 Provision of sound pre-enrichment procedures for
9
1, 2
Lactobacillus, Megasphaera and Pectinatus spp.
R6 PCR based assays for the detection of Lactobacillus, 10-12
1, 2, 4
Megasphaera and Pectinatus spp. in beer. Dissemination
to WP3, WP4 and WP5.
R7 PCR kit prototypes for Lactobacillus, Megasphaera and 12, 15
4
Pectinatus spp. in beer. Dissemination to WP3, WP4 and
WP5.
D8 Matrix for the performance of the experiments and for the 10, 23
1-4, 10
collection of data.
D9 Education programme for technical staff in breweries.
10
1-4, 10
Nature
Dissemination
level2
target3
R
PU
C, B
O
RE/CO
R/C
O
RE/CO
R/C
O
RE/CO
R/C
O
RE/CO
R/C
D
RE/CO
R/C
CO
C
O
RE
R
O
PU
B, F, R
1, 3
R
PU
B, F, R
1, 3
R
PU
B, F, R
all
SC1, 1,
10
SC10
R
O
CO
RE
C
R
O
PU
B, F, R
5
O
PU
D16 Provision of reference material for the method 12-17,
SC10
comparison study.
22-28
R17 Automated fluorogenic on-line detection assays for 16, 23, 28, 2,4, 7, 8
unfiltered process samples.
34
R18 Provision of internal oligonucleotide probes for
18
3
hybridisation.
D19 Training Workshop.
18, 30
2, 4, 10
D20 Reports presented for an extended audience at EBC 18, 30, 36 1-5, 10
meetings.
D21 Scientific and technical articles.
starting 18 1-5, 10
O
PU
A, B,C,
F, CO
R, B
O
CO
C
O
PU
R
O
O
PU
PU
O
PU
R22 Provision of sound pre-enrichment procedures for other
beer spoilage taxa.
D23 Objective data on the intrinsic technical quality of the
PCR methods/kit prototypes developed for the analysis of
beer. Dissemination to WP5 and WP6.
R24 PCR based assays and kit prototypes for the detection of
other spoilage organisms in beer. Dissemination of new
methodology to WP3, WP4 and WP5.
D25 Data on the adaptability of the PCR technology to
brewery QC laboratories. Dissemination to WP6.
D26 Data on the performance and practicability of the
developed PCR methods/kit prototypes under brewery
R10 Data on the suitability of rRNAs and mRNAs as viability
12
indicators of brewery contaminants. Dissemination to
WP6.
R11 Data on the faith of DNA in dead cells of brewery
12
contaminants. Dissemination to WP6.
12 Annual report.
12, 24, 36
D13 Provision of experimental plan for the method 12, 22
comparison study.
D14 Deposition of the test organism set to ECCO culture
12
collection.
D15 On line help.
12
10
19
1, 2
O
RE/CO
B
A, B, F,
R
A, B,
CO, F
R/C
19, 30
SC1, 10
R
PU
A,B,F,R
20
1, 2, 4
D
RE/CO
R/C
21, 34
all
R
PU
A,B,F,R
21, 34
all
R
PU
A,B,F,R
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
conditions: beer samples. Dissemination to WP6.
R27 PCR based assays and kit prototypes for the detection of
21
1, 2, 4
D
RE/CO
R/C
spoilage organisms in unfiltered process samples.
Dissemination to WP3,WP4 and WP5.
R28 Automated fluorogenic on-line assays for detection of
23
2, 4, 7, 8
O
CO
C
beer contaminants.
R29 A user-friendly method for the detection of viable
24
3
R
PU
R,B,F
spoilage organisms in unfiltered process samples.
Dissemination to WP6.
D30 Comparative data on the on line detection assays,
29
2, 7, 8
R
PU
A,B,F,R
standard PCR and electrophoresis.
R31 High-throughput assay for detection of amplicons of
30
3
R
PU
B, F, R
brewery contaminants based on PCR-ELISA.
D32 Objective data on the intrinsic technical quality of the
30
SC1, 10
R
PU
A,B,F,R
PCR methods/kit prototypes developed for the analysis of
unfiltered process samples. Dissemination to WP5 and
WP6.
R33 Comparative data on the operational characteristics of on34
2, 3, 7, 8
R
PU
A,B,F,R
line PCR-assays, PCR-ELISA and electrophoresis to
assist breweries in the selection of a suitable detection
method for PCR amplicons. Dissemination to WP6.
D34 Data on the performance and practicability of the
34
all
R
PU
A,B,F,R
developed PCR methods/kit prototypes under brewery
conditions: unfiltered process samples. Dissemination to
WP6.
D35 Analytical Standards Methods.
36
2, 3, 4, 10
O
PU
A,B
36 Final report.
36-40
all
R
CO
C
D37 Special Symposium on the application of PCR 36-42
1
O
PU
A,B,F,
technology.
CO
1
R=R & D phase deliverable; D=Demonstration phase deliverable; no mark: R & D and Demonstration phase deliverable
2
PU=public, RE=restricted to a group specified by the consortium (including EC services), CO=confidential, only for the
members of the consortium (including EC services)
3
A=authorities; B=brewing industry; C=consortium; CO=consumers; F=other food industry; R=research
11
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Estimated timetable of workpackages (detailed Timetable: See Annex 1).
YEAR 1
Workpackage
WP1
WP2
1
3
5
7
9
11
13
15
YEAR 2
Months
17
19
YEAR 3
21
23
25
27
29
31
33
35
PCR based detection methods for spoilage organisms in bright beer
36- Partners
42
P1 (P2-4, P10)
P3 (P1, P2,P4)
PCR based detection methods for spoilage organisms in process samples
WP3
P4 (P2, P3,
P7, P8)
P10 (P1, SC1,
SC10)
P2 (P1-10,
SC1)
P5 (P1-4, P10)
Development and application of improved detection methods for PCR products
WP4
Method comparison study
WP5
Evaluation in industry
WP6
Dissemination of knowledge
Milestones
1
2
4
5
6
8
3
7
9
R & D phase
10
11
Demo phase
Meetings and reporting

P; partner SC; subcontractor
PM; Project Management Group

R & D wg: R & D Working Group
12
DEMO wg: Demo Working Group
 
PM
R&D wg
DEMO wg
IPF
Reports
IPF; Industrial Platform
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Figure 2. The management structure.
DEMONSTRATION PHASE
RESEARCH & DEVELOPMENT PHASE
FEED -BACK
PCR METHODS,
WP1:
PCR
based
detection methods for
spoilage organisms in
bright beer
Partner: 1, 2, 3, 4, 10
KIT PROTOTYPES
WP2: PCR based detection
methods
for
spoilage
organisms in unfiltered
process samples
Partner: 1, 2, 3, 4
WP4:Method comparison
study
Partner: 10, (1)
Subcontractor: 1, 10
WP5: Industry evaluation
Partner: 1-10
Subcontractor: 1
INFORMATION
PCR PROTOCOLS
WP3:
Development
and
application of improved detection
methods for PCR amplicons
Partner: 2, 3, 4, 7, 8
WP6: Dissemination of knowledge
Partner: 1, 2, 3, 4, 5, 10
INFORMATION
13
BREWPROC
2.3
Technical annex
Project proposal Nr QLRT-1999-31251
Workpackage descriptions
Workpackage number: 1
Phase: Research
Start date: 1 month
Completion date: 20 month
Partners responsible: 1, 2-4, 10
Person months (per partner) and total: 16 (P1), 15 (P2), 5 (P3), 14 (P4), 2 (P10): 52 (total)
Objectives
 Development and establishment of robust PCR based assays capable of detecting low amounts (< 10 cfu per 500 ml) of
viable spoilage microorganisms in bright beer in 1-2 days.
 Development of kit prototypes for PCR detection of spoilage microorganisms in bright beer.
Methodology and study materials
Task 1.1 Comparison of existing PCR assays. P3 will compare various simple methods described for the preparation of
PCR amplifiable DNA from beer samples in terms of their sensitivity (cfu/ml), reliability and practicality using spiked and
non-spiked beers (200-400 PCR analyses). P10 will prepare the study material (a minimum of 3 beer types spiked with 3-5
levels of 3-4 test organisms). The resulting DNA extracts will be analysed under identical PCR and post-PCR conditions.
The specificity and sensitivity of the alternative primer sets available for Lactobacillus, Megasphaera and Pectinatus spp.
will be compared using pure cultures of target and non-target organisms. P3 will report results to the consortium who selects
the best practice methods for further studies.
Task 1.2 Oligonucleotide design and PCR optimisation. P1, P2 and P4 will design new PCR primers for those beer
spoilage taxa for which primers are not yet available (e.g. acetic acid bacteria). The target sequences will be sequenced
when necessary. The PCRs will be internally standardised and optimised. The specificity and sensitivity of the reactions will
be determined with pure cultures of appropriate strains.
Task 1.3 Detection of new target organisms in beer by PCR.. P1, P2 and P4 will optimise the pre-PCR steps (including
pre-enrichment, task 1.4) for the new target organisms. Sensitivity of the detection assays will be studied with spiked beers.
Task 1.4 Pre-enrichment. P1 and P2 will compare various growth based and physical methods for enrichment to detect less
than 10 cells per 500 ml beer. For enrichment cultivation, various methods (forcing, enrichment after membrane filtration)
and media (selective and non-selective) will be tested using cultivation, PCR and/or turbidometry. Considerations are
detection time, ease of use and the effect of medium components and background microflora on PCR results. Not less than
two strains per species will be tested. Minimum enrichment times that will ensure a positive PCR detection of low numbers
of the target organisms (2-4 strains/species) will be determined. Beer samples will be inoculated with various levels of test
cultures and analysed by PCR and/or by cultivation after different time periods. First, the effect of various stress factors on
the growth of the contaminants will be studied. Physical methods to be assessed include for example magnetic separation.
Task 1.5 Formulation of PCR kit prototypes. P4 will evaluate the established PCR methods, and then exploit them to
develop PCR kit prototypes for beer samples. Special emphasis will be put on the design of controls and on the shelf-life of
the components and on the user-friendliness of the prototype. The sample preparation procedures will be automated as far as
possible, and together with the specific primer sets, a modular kit system will be developed.
Deliverables (Type of deliverable)
1. Comparable data on the performance of the simple PCR based assays existing for beer spoilage bacteria.
Dissemination to WP2, WP3 and WP6. (PU)
2. Provision of internal standards for PCR reactions specific to Lactobacillus, Megasphaera and Pectinatus spp..
Dissemination to WP2 and WP3. (RE/CO)
3. Provision of sound pre-enrichment procedures for Lactobacillus, Megasphaera and Pectinatus spp. (RE/CO)
4. PCR based assays and kit prototypes for the detection of Lactobacillus, Megasphaera and Pectinatus spp. in beer.
Dissemination of new methodology to WP3 and to WP4 and WP5 for demonstration. (RE/CO)
5. Provision of internally standardised PCR reactions specific to other beer spoilage taxa. Dissemination to WP2
and WP3. (RE/CO)
6. Provision of sound pre-enrichment procedures for other beer spoilage taxa. (RE/CO)
7. PCR based assays and kit prototypes for the detection of other spoilage organisms in beer. Dissemination to
WP3, WP4 and WP5. (RE/CO)
Milestones
10 months: Reliable PCR based assays and kit prototypes for testing beer for the presence of Lactobacillus, Megasphaera
and Pectinatus spp. successfully established .
20 months: Reliable PCR based assays and kit prototypes for testing beer for the presence of other beer spoilage taxa
successfully established.
14
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Workpackage number: 2
Phase: Research
Start date: 1 month
Completion date: 21 month
Partners responsible: P3, P1, P2, P4
Person months (per partner) and total: 24 (P3), 18 (P1), 15 (P2), 16 (P4): 73 (total)
Objectives
 Development of robust PCR based methods for detecting low levels (< 102 cfu/2·107 yeast cells) of spoilage microorganisms in unfiltered process samples.
 Development of RT-PCR based methods capable of detecting of viable spoilage micro-organisms in unfiltered process
samples.
 Development of kit prototypes for PCR detection of spoilage micro-organisms in unfiltered process samples.
Methodology and study materials
Task 2.1 Design of process contaminant specific primers and PCR optimisation. P1, P2 and P4 will design and validate
specific primers for primary contaminants of the brewing process. The target organisms include e.g. pediococci and wild
yeasts. False negative interpretations will be obviated by internal standardisation of the reactions. The new primer sets will
be also exploited for the detection of the same organisms in bright beer (WP1).
Task 2.2 Detection of process contaminants by RT-PCR. P1 and P3 will investigate the suitability of reverse transcriptase
(RT)-PCR for the selective detection of viable cells. Both translation elongation factor messenger RNA (TEf-mRNA) and
rRNA will be used as primer targets. The sequencing of the areas of interest may be necessary. The relationship between the
detection of RNA by RT-PCR and viability will be studied in cells killed by various treatment which are relevant to brewing
process (e.g. heat, ethanol, disinfectants). Northern blotting will be used as a reference method. As a comparison, the
disappearance of DNA in similarly treated cells will be studied by respective DNA-PCRs. Test organisms will be
representative of the major spoilage species. Later, the effect of matrix (beer/growth medium) and the growth state and
environmental conditions on the expression of the targeted RNA species will be studied. With a successful implementation
of RT-PCR to viability assessment, P3 will optimise the method for the detection of the target organisms in unfiltered
process samples by adapting published methods.
Task 2.3 Simple nucleic acid isolation and Task 2.4 Treatment of unfiltered samples for PCR. The research will be focused
on the development of easy and effective procedures for the preparation of PCR amplifiable DNA from unfiltered process
samples. P1 and P2 will compare different physical and microbiological enrichment techniques to maximise the sensitivity,
rapidity and simplicity of the pre-nucleic acid isolation step. These will include, for example, dielectrophoresis, magnetic
separation, centrifugation, filtration, cultivation enrichment and different combinations of them. The sensitivity and time
requirements of the techniques will be studied by analysing unfiltered samples spiked with various levels of test cultures.
Identical DNA isolation methods and PCR conditions will be used when possible to obtain comparable data. Different
simple DNA extraction procedures and commercial kits including the methods used in WP1 will be screened for DNA
isolation (P1, P2, P4). The sensitivity of the isolation protocols will be assessed with the pure cultures of gram-negative and
-positive process contaminants. The second approach will be the use of nested PCR in combination with simple sample
treatment methods.
Task 2.5 Development of PCR kit prototypes for unfiltered samples. P4 will exploit the developed sample preparation
methods and oligonucleotide primers to formulate PCR kit prototypes for unfiltered process samples.
Deliverables (Type of deliverable)
1. Provision of internally standardised PCR reactions specific to process contaminating organisms. Dissemination
to WP1 and WP3. (RE/CO)
2. Data on the suitability of rRNAs and mRNAs as viability indicators of brewery contaminants. Dissemination to
WP6. (PU)
3. Data on the faith of DNA in dead cells of brewery contaminants. Dissemination to WP6. (PU)
4. PCR based assays and kit prototypes for the detection of spoilage organisms in unfiltered process samples.
Dissemination of new methodology to WP3 and to WP4 and WP5 for demonstration. (RE/CO)
5. A user-friendly method for the detection of viable spoilage organisms in unfiltered process samples established.
Dissemination to WP6. (PU)
Milestones
12 months: Suitability of RT-PCR detection of rRNA and mRNA for viability assessment successfully evaluated.
21 months: PCR based assays and kit prototypes for spoilage organisms in unfiltered process samples successfully
developed.
24 months: RT-PCR based assays for viable spoilage organisms in unfiltered process samples successfully developed.
15
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Workpackage number: 3
Phase: Research
Start date: 1 month
Completion date: 34 month
Partners responsible: P4, P2, P3, P7, P8
Person months per partner and total: 12 (P2), 27 (P3), 14 (P4), 12 (P7), 12 (P8): 77 (total)
Objectives
The over-all aim is to improve and facilitate the detection of PCR products in such a way that sample throughput, the
interpretation of results, quantification possibilities and work safety are enhanced. This will be achieved by
 developing automated fluorogenic on-line assays to detect and quantify target micro-organisms
 developing a low cost hybridisation assay to detect multiple target organisms in a single colorimetric assay.
Methodology and study materials
Task 3.1 Development and application of on-line detection assays based on fluorogenic probes. The 5´-nuclease and the
Hyb-probe assay will be investigated for the fluorogenic on-line detection of PCR products. The former is based on the
cleavage of a fluorogenic probe by 5´-exonuclease activity of Taq DNA polymerase whereas the latter relies on the
adjoining annealing of two probes. P2, P4 , P7 and P8 will investigate the primers arising from WP1 and WP2 for their
compatibility with the two detection formats. Design of new primes may be necessary. Internal fluorogenic DNA probes
will be targeted within the sequences delineated by the specific primers, and the PCR conditions will be optimised for the
fluorogenic detection. The specificity of the detection assays will be determined using a representative set of target and
reference organisms. Both laboratory cultures and spiked brewery samples will be employed for the sensitivity assessment.
The sample preparation for PCR will be initially by methods arising from WP1 and WP2. When found necessary, the prePCR steps will be optimised with the new assays. Finally, the industrial partners 7 and 8 will test the applicability of the
assays to the analysis of actual brewery samples. Thermocycling and the analysis of PCR products will be carried out using
LightCycler (Roche) or TaqMan system (PE Applied Biosystems). Electrophoretic methods will be used as references.
Task 3.2 Development and application of detection assays based on microtitre plate hybridisation. P3 will develop
oligonucleotide probe hybridisation based on the methods developed by Partner 1 in previous studies and others. Internal
oligonucleotide probes will be designed for the PCR amplicons developed in WP1 and WP2. A selection of probes specific
for e.g. Lactobacillus spp., wild yeasts and other beer spoilage organisms will be synthesised by a commercial laboratory
and covalently bound to microtitre plates. The PCR methods from WP1 and WP2 will be used to generate PCR amplicons
of the selected beer spoilage organisms. The amplicons will be analysed by sandwich hybridisation with two
oligonucleotide probes. The capture probe will be covalently linked to the microtitre plate. The detection probe will be
labelled with biotin. Hybrids of oligonuleotide probes and PCR amplicons will be detected by incubation with avidin
conjugated enzyme and a chromogenic substrate. The resulting colorimetric change will be interpreted by an OD
measurement. The specificity and the sensitivity of the assay in comparison with Southern hybridisation and gel
electrophoresis will be elucidated using purified PCR products, pure cultures and spiked brewery samples, as appropriate.
Task 3.3 Comparison of different detection methods for PCR products. P2, P3, P7 and P8 will compare the developed
assays with the assistance of Partner 3. Considerations are ease of use and adoption, speed, sensitivity (cfu/ml), sampling
capacity, affordability and costs of operation. A representative set of spiked samples will be analysed in parallel with the
developed methods and gel electrophoresis. The results will be reported and passed to WP6 for dissemination. The number
of methods ultimately evaluated will depend on the technical advances achieved.
Deliverables (Type of deliverable)
1. Automated fluorogenic on-line assays for detection of beer contaminants. (CO)
2. Provision of internal oligonucleotide probes for hybridisation. (PU)
3. Automated fluorogenic on-line detection assays for unfiltered process samples. (CO)
4. High-throughput assay for detection of amplicons of brewery contaminants based on PCR-ELISA. (PU)
5. Comparative data on the on-line detection assays and electrophoresis. (PU)
6. Comparative data on the operational characteristics of on-line PCR-assays, PCR-ELISA and electrophoresis to
assist breweries in the selection of a suitable detection method for PCR amplicons. Dissemination to WP6. (PU)
Milestones
34 months: Automated on-line systems and PCR-ELISA assays developed and successfully implemented.
16
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Workpackage number: 4
Phase: Demonstration
Start date: 10 months
Completion date: 30 months
Partners responsible: P10, P1, SC1, SC10
Person months per partner and total: 1 (P1), 4 (SC1), 21 (P10), 4 (SC10): 30 (total)
Objectives
The aim of this workpackage is to establish objectively the intrinsic technical quality of the PCR based methods/kit
prototypes arising from the R & D phase of the project in comparison with reference methods through use of artificially
contaminated brewery samples. Considerations are: specificity, limit of detection, relative accuracy and user-friendliness.
Methodology and study materials
The tasks of this WP will be performed in two phases.
Task 4.1 Experimental design. A study protocol for the method comparison will be designed by a statistician (SC1) with
the assistance of research partners (P1, P10, SC10). It will detail the number of samples and replicates to be tested, the
microbial composition of samples including target and competing micro-organisms and their respective cell counts and
viability, reference methods and the method attributes under study. A detailed test procedure will also be provided. The
number of methods evaluated will depend on the success of the methodology development work carried out in the R & D
phase.
Task 4.2 Preparation of reference material. An independent laboratory (SC10) will collect a comprehensive set of
brewery contaminants from other partners for the preparation of the reference material. Samples will be prepared by
contaminating brewery samples (beer, unfiltered process samples) according to the study protocol. The samples will vary in
microbial composition and inoculation level. The reference material will be delivered by courier to the expert laboratory
(P10) carrying out the method comparison. The test organisms will be added to ECCO culture collection (SC10).
Task 4.3 Performance of the method comparison study. The study protocol will be verified in a preliminary trial. The
comparison of the PCR methods/kit prototypes against the reference methods outlined in EBC Analytica Microbiologica or
in MEBAK will be performed through independent testing. The same reference samples will be analysed in parallel with the
reference and PCR procedure. Each PCR method will be scored on limit of detection, specificity, relative accuracy and ease
of use. Broadly, the limit of detection and specificity will be determined by analysing samples containing various levels of
target organisms and mixtures of target and competing microbes. Relative accuracy will be defined through comparison of
the PCR results to the results obtained using the reference methods.
Task 4.4 Analysis and reporting of results. P10 will compile the results and deliver them to SC1 for a statistical analysis.
Progress and final reports will be prepared by the expert laboratory in co-operation with the statistical expert, and they are
scheduled at the end of both demonstration phases. The reports are passed to WP5 for reviewing and to WP6 for
dissemination.
Deliverables (Type of deliverable)
1. Provision of experimental plan for the method comparison study. (RE)
2. Provision of reference material for the method comparison study. (PU)
3. Deposition of the test organism set to ECCO culture collection. (PU)
4. Objective data on the intrinsic technical quality of the PCR methods/kit prototypes developed for the analysis of
beer. Dissemination to WP5 and WP6. (PU)
5. Objective data on the intrinsic technical quality of the PCR methods/kit prototypes developed for the analysis of
unfiltered process samples. Dissemination to WP5 and WP6. (PU)
Milestones
30 months: Intrinsic technical quality of the developed PCR methods/kit prototypes in the analysis of artificially
contaminated beer objectively demonstrated.
30 months: Intrinsic technical quality of the developed PCR methods/kit prototypes in the analysis of artificially
contaminated process samples objectively demonstrated.
17
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Workpackage number: 5
Phase: Demonstration
Start date: 8 month
Completion date: 34 month
Partners responsible: P2, P1, SC1, P3-10
Person months per partner and total: 7 (P1), 6 (SC1), 12 (P2), 4 (P3), 7 (P4), 11 (P5), 8 (P6-8), 12 (P9), 5 (P10): 88
(total)
Objectives
 To demonstrate the transferability of the PCR technology to brewery quality control environment.
 To demonstrate the practicability and the operational characteristics of the PCR methods/kit prototypes developed in
the R&D phase under typical application conditions.
 To demonstrate the benefits of using PCR in routine monitoring of brewery samples.
 To improve the acceptance of the PCR methods as a microbial testing application in food industry.
Methodology and study materials
The tasks in this WP will be performed in two phases:
Task 5.1 Organisation of the study. Research partners 1-4 and 10 will plan and organise the study in collaboration with a
statistician (SC1). An experimental plan outlining minimum testing requirements, sample types under study, time schedule
and reference methods will be prepared. Detailed instructions for performing the PCR analyses and questionnaires for
recording observations, results and deviations will be also provided. A training concept for the technical staff in breweries
will be established.
Task 5.2 Education of technical staff in breweries. Brewery QC laboratories will be provided with information about the
required equipmentation and laboratory arrangements. P1-4 will share the responsibility of educating the brewery technical
staff to work with molecular biological techniques and with the developed PCR methods. The basic training will be
arranged in the research laboratories followed by an adoption period in the breweries during which the staff will practice the
PCR testing with spiked brewery samples.
Task 5.3 Evaluation of the PCR methods in brewery QC laboratories. QC samples will be analysed in parallel with the
PCR methods and routine methods. The number of PCR methods evaluated will depend on the advances made in the R&D
part. Negative and positive control samples will be supplied at regular intervals by P10. The use of PCR in breweries will
last over two periods of up to 6 months each. The actual time needed will depend on the QC practices of the individual
breweries. The first phase will demonstrate the methods arising from WP1 after 10 months, the second phase will use the
methods available from WP1 and WP2 after 20 months. All work will be overseen locally by brewery QA management and
monitored by the research partners. Breweries will also have a task to estimate financial benefits following from the use of
PCR in QC. The suitability of the PCR technology to a QC environment and practicability and benefits of the PCR methods
will be studied via questionnaires and interviews on completion of the testing phases. Considerations will be the ease of use
and adoption by the technical staff, work safety related issues, sampling capacity, suitability to laboratory routines,
sensitivity to DNA contaminations, and similar attributes.
Task 5.4 Analysis and reporting of results. The results (analysis data, questionnaires) will be collected, analysed and
reported by SC1 and P1-4 after the testing phases. On completion, a final report covering the results obtained in WP4 and
WP5 will be prepared. The reports will be passed to WP6 for dissemination.
Deliverables (Type of deliverable)
1. Matrix for the performance of the experiments and for the collection of data. (RE)
2. Education programme for technical staff in breweries. (PU)
3. Data on the adaptability of the PCR technology to brewery QC laboratories. Dissemination to WP6. (PU)
4. Data on the performance and practicability of the developed PCR methods/kit prototypes under brewery
conditions: beer samples. Dissemination to WP6. (PU)
5. Data on the performance and practicability of the developed PCR methods/kit prototypes under brewery
conditions: unfiltered process samples. Dissemination to WP6. (PU)
Milestones
34 months: Adaptability of the PCR technology to brewery QC laboratories successfully demonstrated.
34 months: Technical and practical viability of the developed PCR methods/kit prototypes successfully demonstrated.
34 months: Real advantages of using PCR in routine monitoring of brewery samples successfully demonstrated.
18
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Workpackage number: 6
Phase: Demonstration
Start date: 1 month
Completion date: 42 month
Partners responsible: 5, 1-4, 10
Person months per partner and total: 7 (P1), 6 (P2), 5 (P3), 3 (P4), 10 (P5), 10 (P10): 41 (total)
Objectives
The objective of this workpackage is to ensure full awareness and therefore opportunity of implementation and exploitation
of the research and demonstration outputs of the project. Sector targets for dissemination are academic and technical
research institutes, production companies, consumers and authorities. Personnel targets for dissemination will span senior
and technical management through to production personnel and supervising authorities.
Methodology and study materials
The workpackage comprises the following tasks:
6.1 Dissemination to the European brewing industry and to an extended audience
6.2 Preparing of reports, articles and publications
6.3 Organising training workshops



Dissemination of the research and demonstration outputs of the project is carried out by using the existing network of
the European Brewery Convention (EBC), which covers practically all the European breweries. Meetings and symposia
targeted at different groups are organised during the project including meetings of the EBC Detection of Contaminants
Subgroup, the EBC Brewing Science Group, the EBC Analysis Committee and EBC Congresses. A separate
Symposium to an extended audience dealing with the research results and implementation experiences obtained during
the project is arranged after the project by P1. Web pages are constructed with linkages to EU officials, to the EBC, to
production companies and research institutes (P5). Standard methods are incorporated into the EBC Analytica
Microbiologica (P4, P5), IOB Methods of Analysis (P3, P10) and MEBAK (P2).
Progress reports are produced for distribution at the meetings. Scientific results are published in relevant journals
according to agreement between the partners. Articles concerning experience obtained from industrial use of the
technology are published in relevant technical journals. Journals used for publication are those dealing with brewing
research but also more generally food, beverages and microbiological methods.
Training workshops for end users are constructed and organised by P2, P4, and P10.
Deliverables (Type of deliverable)
1. Analytical Standard Methods. (PU)
2. Scientific and technical articles. (PU)
3. Reports presented for an extended audience at EBC meetings. (PU)
4. Training Workshops. (PU)
5. On line help. (PU)
6. Special Symposium on the application of PCR technology. (PU)
Milestones
12 months: On-line information of the technology including list of publications.
36 months: Publication of standard method(s) in EBC Analytica Microbiologica, IOB Methods of Analysis and in MEBAK.
42 months: Special Symposium devoted to the application of PCR technology for the brewing industry and extended
audience.
19
BREWPROC
3.
Technical annex
Project proposal Nr QLRT-1999-31251
ROLE OF PARTICIPANTS
Partner 1: VTT Biotechnology (former VTT Biotechnology and Food Research), P.O. Box 1500
(Tietotie 2), FIN-02044 VTT, Finland
Scientific team: Team leader: Dr. (Agr. & For.) Auli Haikara, Research Scientists: Dr. (Agr. & For.)
Erna Storgårds, MSc (Microbiology) Riikka Juvonen, one scientist to be appointed.
Contractual links to other participants: Subcontractor 1, VTT Internal Services
Objectives: Co-ordinator of the project, responsible of WP1. The R & D role is to develop robust,
sensitive and reliable PCR based procedures for the detection of spoilage bacteria in beer and in
process samples and to investigate the suitability of RT-PCR for the viability assessment of brewery
contaminants. The demonstration role is to assist in the planning, organisation, performance and
reporting of the demonstration tasks and to participate in the dissemination of the project outputs.
Workplan: WP1 (16 months): Tasks 1.2 (3), 1.3 (3) and 1.4 (10). Internal standards will be
constructed for the best practice PCRs to avoid false negative results, and the PCR conditions will be
optimised for detection in beer. The sensitivity and specificity of the standardised PCRs will be studied
using pure DNA and spiked beer samples. To detect low levels of bacteria in beer by PCR, growth
based pre-enrichment procedures will be developed using impedimetry, turbidometry, cultivation and
PCR as research tools. First, different methods (e.g. forcing, pre-growing after beer filtration) and
media (e.g. SMMP, MRS, EC-MRS, C-NBB) will be compared ( 2 strains/species) to find a protocol
that allows good growth, is easy to apply and is compatible with the specific PCRs. Subsequently,
minimum enrichment times needed for the detection of less than 10 cfu per 500 ml will be determined.
The effect of different stress factors (e.g. heat, chemicals) on the growth of spoilage bacteria will be
investigated in the preliminary studies.
New PCR based detection strategies for beer spoilage bacteria will be developed and evaluated in cooperation with P2. As most beer samples are free from contaminating organisms, unspecific PCR
reactions will be set up to pre-screen samples for the presence of any bacteria. For the identification of
the detected bacteria, species- and group specific PCRs (multiplex/single) will be designed. In another
approach, multiplex PCRs incorporating primer pairs for various beer spoilage organisms will be
developed to simultaneously identify several spoilage organisms in beer. Shortly, the development
work will include the design, optimisation and validation (specificity, sensitivity) of new specific PCRs
with the incorporation of internal standards and the optimisation of the pre-PCR steps (pre-enrichment,
DNA isolation, inhibitor removal) for sensitivity, rapidity and robustness. The exact workplan will
depend on the outcomes of the research work which are not fully predictable.
WP2 (18 months): Task 2.1 (2), 2.3 (2) and 2.4 (10). In co-operation with P2, the team will develop
PCR based detection assays for spoilage bacteria in unfiltered process samples (pitching yeast,
fermenting wort, unfiltered beer). Internally standardised PCR reactions will be designed for process
contaminants and optimised in regard to sensitivity and specificity. The effect of yeast DNA on the
PCR detection of specific DNA sequences will be studied. To maximise sensitivity, rapidity and
simplicity of sample pre-treatment, enrichment cultivation and different separation and concentration
techniques (e.g. dielectrophoresis, magnetic separation of cells or their DNA, centrifugation and
filtration) will be compared. Simplified procedures and commercial kits will be investigated for DNA
isolation. The most promising sample treatment methods will be further optimised. In addition, the use
of nested PCR for improving the assay sensitivity will be investigated.
Task 2.2 (4).The potential of rRNA detection by RT-PCR for viability assessment will be evaluated.
The cells (a set of major spoilage species) will be killed by chemicals/treatments relevant to brewing.
The fate of rRNA and DNA in killed cells will be studied by RT-PCR and PCR as a function time. The
effect of the growth phase and environmental conditions on the expression of rRNA will be
investigated by RT-PCR and Northern blotting.
WP4 (1 months). Task 4.1 (1). The team will consult the other partners involved in microbiology
and/or methodology related matters and provide SC10 with microbial strains for deposition and
preparation of reference material.
WP5 (7 months). Task 5.1 (1), 5.2 (2), 5.3 (2), 5.4 (2). The team will assist SC10 in the statistical
design of experiments by providing the methodology and microbiology related expertise. Other
20
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
demonstration activities will be organised and reported together with other partners involved. Team
will be responsible of consultation, training, interviewing and surveillance of industry evaluation at P6.
WP6 (7 months): Task 6.1 (4), Task 6.2 (3). The team will be responsible of contacts to EBC including
Brewing Science Group, the EBC Analysis Committee and the EBC Detection of Contaminants
Subgroup. Two of the team members are affiliated to EBC Groups and Subcommittees. Furthermore,
the EBC Detection of Contaminants Subgroup is chaired by Dr. Storgårds. Reports will be produced on
key results, and in agreement with other partners the project outputs will be published in scientific
journals. The team will be responsible of arranging the End Symposium on the application of the PCR
technology.
Deliverables: See Table 3.
Subcontractor 1: Technical Research Centre of Finland, VTT, Internal Services, B. O. Box 2100
02044 VTT, Finland
Scientific team: Leila Peltoniemi-Laajanen, Planning Officer, M.Sc. (Stat.)
Contractual links to other participants: Subcontractor to Partner 1, VTT Biotechnology
Objectives: Responsible for the statistical design of the method comparison study (WP4) and industry
evaluation (WP5) and for the statistical analysis of the results.
Workplan: WP4 (4 months): Task 4.1 (2), 4.4 (2). Principal responsibility in the test planning. The
partner will define minimum testing requirements including the number of samples and replicates, the
composition of the samples and parameters to be determined. In the statistical analysis of results the
limits of sensitivity will be specified and the comparison of PCR based methods to reference methods
will be statistically tested. Results of the study will be reported in collaboration with P10.
WP5 (6 months): Task 5.1 (3), 5.2 (3). Experimental design in collaboration with research partners for
demonstrating the technical quality of the PCR methods and kit prototypes in the analysis of actual
brewery samples. Participation in designing questionnaire to users of PCR methods. Statistical analysis
to evaluate PCR methods in comparison with the established methods to improve the acceptance of
PCR methods.
Deliverables: See Table 3.
Partner 2: TU München-Weihenstephan (TUM), Lehrstuhl für Technologie der Brauerei II, Alte
Akademie 3, 85350 Freising-Weihenstephan, Germany.
Scientific team: Team leader: Prof. Dr. Eberhard Geiger, Scientist: Thomas Schuhbeck, Master of
Science in Technology
Contractual links to other participants: No.
Objectives: TUM will contribute to the design of oligonucleotides for beer spoilage bacteria and to the
optimisation of the PCR detection systems and sample preparation methods for filtered and unfiltered
beer (WP1 and 2). Further, on line assays will be developed and implemented for the detection of beer
spoilage bacteria. The different detection methods developed for the analysis of PCR products will be
compared and evaluated concerning their sensitivity and practicability (WP3). The team will be
responsible of WP5 and will participate in the organisation, realisation and reporting of the industry
evaluation. Furthermore, the team will participate in the dissemination of the project outputs (WP6).
Workplan: WP1 (15 months). Task 1.2 (4). After DNA database searches, primers and probes will be
designed for beer spoilage bacteria and tested concerning their specificity and sensitivity. In the
experiments, pure DNA isolated from appropriate reference and target strains will be employed.
Task 1.3 (4). The designed primers and probes will be tested for the detection of the targeted organisms
in beer using artificially contaminated beer samples. PCR conditions and sample preparation methods
will be optimised for the detection in beer. Subsequently, the sensitivity of the PCR detection systems
will be determined.
Task 1.4 (7). In order to improve sensitivity, several microbiologic pre-enrichment methods (e.g. usage
of different culture media and their optimisation) and physical methods (e.g. filtration, centrifugation,
magnetic beads etc.) will be tested and optimised. The method emerging to be the best will be
optimised to a minimum of time need and user-friendliness.
WP2 (15 months). Task 2.1 (3). After a DNA database search, primers and probes will be designed for
the detection of process contaminating bacteria and tested with DNA isolated from appropriate
21
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
reference strains (see task 1.2). In model experiments, the influence of DNA from brewer's yeast on
the detection of the target organisms will be investigated.
Task 2.3 (4). In order to establish a suitable DNA isolation method, the contaminant cells in yeast
containing samples will be collected quantitatively applying different separation systems. For DNA
extraction, newly developed isolation methods and commercial kits will be used.
Task 2.4 (8). To enhance sensitivity of detection systems, pre-enrichment methods to suppress growth
of yeasts and to support propagation of target organisms will be verified. A minimum of enrichment
time is aimed.
WP3 (12 months). Task 3.1 (6). The compatibility of the developed oligonucleotides (WP1, 2) with the
on-line systems will be studied. When necessary, new primers and probes will be designed for beer
spoilage bacteria. The PCR detection systems will be optimised for on line detection and applied for the
detection of the target organisms. Further, multiplex systems and internal control will be developed
and tested with target DNA.
Task 3.3 (6). The developed and optimised detection systems (e.g. gel electrophoresis, PCR-ELISA and
on line systems) will be compared concerning their practicability and sensitivity. The experiments will
be performed using artificially contaminated samples.
WP5 (12 months). Task 5.1 (2). The study will be organised and planned in collaboration with other
research partners and a statistical expert. The experimental plan outlining testing requirements, sample
types, time schedule and reference methods, the questionnaires as well as training programmes for
breweries´ technical staff will be produced.
Task 5.2 (2). The QC laboratories will be provided with information about the required laboratory
arrangements and equipmentation. The technical staff in breweries will be intensively trained. The
assembly of the instruments and the first experiments will be supervised.
Task 5.3 (4). The team will offer technical support in case of any trouble. The technical staff in
breweries will be interviewed to find out the benefits of PCR in QC laboratories. Questionnaires are
will also be used for this purpose. The analysis results will be collected.
WP 5.4 (4). Suitability and practicability of PCR based methods will be evaluated on the basis of
questionnaires, analysis results and interviews. Reports will be produced in collaboration with other
partners.
WP6 (6 months).Task 6.1 (2). The resulting PCR methods will be written as standard methods to
MEBAK. In different symposia, theoretical backgrounds and benefits of using PCR methods in
brewery QC will be explained. In education, PCR methods as a means of QC method in breweries will
be taken into standard education.
Task 6.2 (2). Reports and articles will be published in relevant literature to achieve a preferably high
level of awareness.
Task 6.3 (2). Workshops will be planned and organised together with partner 4 to familiarise and train
the end users of PCR based systems.
Deliverables: See Table 3.
Partner 3: Brewing Research International (BRi), Lyttel Hall, Nutfield, Surrey, RH1 4HY, United
Kingdom.
Scientific team: Dr John Hammond (Project Advisor), Dr Samantha Walker (Team leader), Ms Marie
Maugeuret (Microbial and Molecular Biologist)
Contractual links to other participants: No.
Objectives: BRi will be responsible for evaluating the various PCR methods for the detection of
spoilage bacteria in beer to define the best practice procedure for further studies. We will play a key
role in the development of novel assays for the detection of brewing contaminants in beer and in
process samples. Firstly, RT-PCR methods will be developed. The main advantage of this technique is
that only viable cells are detected. Secondly, colorimetric microplate hybridisation assays will be
established to facilitate post-PCR analysis. We are also responsible for the planning of the QC trials in
UK breweries and for the education of the relevant personnel as well as in disseminating the project
outputs.
Workplan: WP1 (5 months), Task 1.1. The PCR assays and primer pairs existing for detection of
spoilage bacteria in bright beer will be defined in regard to their sensitivity (cfu/ml), specificity,
22
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
reliability and ease of use using artificially contaminated beers provided by P10. A minimum of three
types of beer containing approx. 101-108 cells of Megasphaera, Pectinatus or Lactobacillus will be
analysed. Total number of PCR analyses will lie between 200 and 400.
WP2 (24 months), Task 2.2. The team is mainly responsible for development and evaluation of RTPCR methods for detecting viable contaminants in process samples. The suitability of translation
elongation factor mRNA as viability indicators for rapid detection of bacteria will be defined. This may
necessitate sequencing of areas of interest to establish areas suitable for probing. In the first instance,
the methodology will be evaluated with major spoilage species. Tests will be conducted to determine
the effects of cell death, growth phase (lag, exponential and stationary) and growth conditions
(aerobic/anaerobic, beer or nutrient medium) on the detection of RNA by RT-PCR. Northern blotting
will be used as a reference method. If the primary evaluations are successful, a procedure for the
detection of spoilage organisms in unfiltered process samples will be developed by adapting published
methods.
WP3 (26 months), Task 3.2 (24), Task 3.3 (3). Development of microplate hybridisation techniques to
detect multiple contaminants in a single colorimetric hybridisation assay. Oligonucleotide probe
hybridisation assays will be performed in a microtitre plate format. Internal probes specific for the
identification of the PCR amplicons specific to Lactobacillus, Pediococcus, acetics, Megasphaera and
Pectinatus will be designed. The capture oligonucleotide will be covalently linked to a microtitre plate.
The detection oligonucleotide will be labelled with biotin. The hybrid molecules will be detected by
avidin conjugated with enzyme and a chromogenic substrate. The colour reaction will be measured in a
microplate reader. The specificity of the assay will be tested using species related to the target organism
and other species occurring in beer and process samples as references. The sensitivity of the
hybridisation assay to pure and mixed cultures of these species will be tested at dilutions of 10 to 107
cells ml-1. The microplate hybridisation techniques developed in part 3.2 of this project will be
compared with standard gel electrophoresis and other methods developed during the course of this
project in terms of sensitivity, ease of use, reliability and specificity.
WP5 (4 month), 5.1 (1), 5.2 (1), 5.3 (1), 5.4 (1). We will give assistance in experimental design, design
of questionnaires and writing of test instructions to partners 1, 2, 4 and 10. PCR technology is not
widely established in the UK brewing industry. The team will be responsible for familiarising the UK
participants (P5) with the methodology developed during the R&D phase of this project. The data
generated during the UK brewery QC studies will be collated for statistical analysis and will be
analysed in conjunction with data collated from other European breweries. A report will be prepared
and passed on to WP6 for dissemination.
WP6 (5 months), Task 6.1 (3), 6.2 (2): Standard methods for use of PCR technology for the analysis of
brewery samples will be prepared and submitted to the Institute of Brewing for publication. The team
will also participate in preparing reports, articles and publications to ensure full awareness and
exploitation of the research and demonstration elements of this project both within brewing and related
industries.
Deliverables: See Table 3.
Partner 4: PIKA Weihenstephan GmbH (former Dr. Vogeser D-TECT), Alte Akademie 3, D-85350,
Freising, Germany.
Scientific team: Dr. Gudrun Vogeser (Team leader), Dipl.-Ing. Andreas Scherer, (Engineer/ Scientist).
Contractual links to other participants: No.
Objectives: PIKA Weihenstephan GmbH´s contribution to the R & D part of the project will focus on
the development of PCR on line detection systems and on the establishment and deliverance of PCR
kit prototypes for the tests in the breweries. In the demonstration part, PIKA Weihenstephan GmbH
will help to establish the PCR methods in breweries' process control by participation in the education
programme for breweries' personnel as well as in the performance of workshops.
Workplan: WP 1 (14 months). Task 1.2 (3): For the detection of different beer spoilage microorganisms DNA sequences useable as primer targets for PCR will be chosen by search of DNAdatabases and sequencing of DNA fragments, if necessary. The PCR conditions will be optimised
(temperature profile, time steps, buffer composition), and the detection limits of the PCR reactions will
be measured with dilution series of pure DNA. Possible cross reactions with other organisms will be
23
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
checked. Internal standards for the detection of primary beer spoilage bacteria will be developed.
Whenever possible, the target sequences will be selected to be used for standard PCR as well as for use
with fluorogenic probes needed for the PCR on-line assays. The specificity of primers and probes will
be tested on pure DNA isolated from the appropriate organisms.
Task 1.3 (3). Previously developed DNA extraction methods will be used for the detection of the new
target organisms with the oligonucleotides developed under Task 1.2. The sensitivity of the whole
detection system (sample preparation + PCR) will be determined by measuring dilution series of pure
cultures as well as of mixed cultures. Different types of beer will be used for the experiments.
Task 1.5 (8). Kit prototypes will be developed for both standard and on line PCR systems. To avoid
false negative results, PCR control systems will be established either by using internal controls
(plasmid DNA) or by the detection of DNA sequences generally present in all samples. The procedure
manuals will be developed and optimised in respect to clarity of understanding and ease of use in a
close co-operation with breweries. Besides, the comments from the breweries will be directly
implemented in respect to possible automation and improvements in handling. Different kit designs for
the components concerning package sizes and physical conditions (liquid, frozen, dried) as well as the
outer packing will be produced for the tests in practice. Storage studies for the different components
under defined temperature conditions (room temperature, cooled, frozen) will be also conducted.
WP2 (16 months).Task 2.1 (4). The work will be done accordingly to Task 1.2., except that the focus
for the micro-organisms to be detected lies on wild yeasts. The influence of DNA from brewer's yeast
on the detection limits for the spoilage organisms will be investigated for standard PCR .
Task 2.3 (4). The methods for DNA isolation from beer samples will be adopted or even newly
established due to the presence of brewer's yeast in the samples. Besides the optimisation of previously
developed DNA isolation methods, commercially available extraction kits will be used.
Task 2.5 (8). The work will be done accordingly to WP 1.5., aiming on the development of detection
kits for bacteria and wild yeast from samples containing brewer's yeast.
WP 3 (14 months). Task 3.1 (14). Specific PCR on line assays will be developed from the primers
designed in WPs 1 and 2. The focus will lie on the genus-specific detection of wild yeasts and primary
bacterial contaminants. To reduce the number of PCR reactions needed per sample, unspecific prescreening PCRs will be developed which will detect any genus of bacteria / non-Saccharomyces-yeast
in the process samples. Only samples positive from pre-screening will be checked with the specific
primers. To further reduce the numbers of PCR reactions needed, multiplex PCR systems will be
investigated. The detection limits of multiplex PCRs will be measured with pure DNA and mixed cell
cultures. If not all primers will fit into the multiplex system, new PCR primers and probes will be
chosen and tested in respect of their usability in multiplex PCRs. Positive as well as negative sample
controls (internal and/or external) will be developed.
WP 5 (7 months). Task 5.1 (1). For the organisation of the study the knowledge gained from our
previously performed pilot study together with breweries about PCR methods will be brought in, e.g.
sample preparation, writing of instructions, and preparation/postage of samples.
Task 5.2 (2). In co-operation with partner 2 the personnel of the breweries will be intensively trained,
first in our research laboratories, then in brewery QC laboratories. The assembly of the instruments and
the first experiments in the breweries will be supervised.
Task 5.3 (3). The work in the QC laboratories will be monitored continuously. Technical support as
well as help in troubleshooting will be given via telephone and E-mail – if needed, by personal visits to
the breweries. At the end of the experimental phases, assistance will be given in the collection of results
and construction of the reports in order to get a uniform and consistent final documentation.
Task 5.4 (1). For both experimental phases in the breweries, the results from all participants will be
collected, compared and written down in reports.
WP 6 (3 months). Task 6.1 (1). In active participation in EBC meetings (EBC Congress, EBC
Subcommittee Microbiology and EBC Detection of Contaminants group) as well as in Symposia, the
state of the art on project progress will be published. In our research laboratories the PCR methods and
the performance of the PCR systems will be demonstrated to people from interested European
breweries not participating in this project.
Task 6.2 (1 month). The results gained during the project work will be published continuously in
relevant technical and scientific journals and on congresses/symposia.
24
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Task 6.3 (1 month). Two workshops will be organised and coached during the project in collaboration
with other partners. Also, a standard method for EBC Analytica Microbiologica will be written in a
combined action of the other partners involved.
Partner 5: Scottish Courage Brewing Limited, Technical Support, 160 Canongate, Edinburgh, EH8
8DD, UK.
Scientific team: Dr. Jeff. A. Hodgson (Team leader), Dr. Behnam Taidi (Scientist) and technician (not
yet appointed).
Contractual links to other participants: No.
Objectives: The role of Scottish Courage Ltd. will be to compare the developed PCR methods and kit
prototypes with the routine cultivation methods in a brewery QC laboratory. The partner will be also
responsible for co-ordinating WP6 and carrying out various dissemination activities.
Workplan: WP5 (11 months), Task 5.3. A set of brewery production samples and bottled beer samples
will be analysed in parallel with the PCR methods developed in the R & D phase and routine
cultivation methods. The testing will be performed in two stages. The practicability and performance of
the PCR tests and the benefits following from the use of PCR in routine QC will be assessed.
WP6 (10 months), Task 6.1 (7), 6.2 (3). The partner will be responsible for co-ordinating, progressing
and delivering the dissemination tasks within agreed time scales. Web pages will be constructed and
maintained. Scientific literature will be reviewed and a list of references arising from the project will be
included in the web pages. The partner will also participate in the preparation of standard methods for
the PCR testing of brewery samples to be issued in EBC Analytica Microbiologica.
Deliverables: See Table 3.
Partner 6: Ringnes a.s., P.O. Box 7152 M, N-0307 Oslo, Norway
Scientific team: Team leader: Tore Hage, process development manager for Ringnes a.s. and chairman
of EBC subcommittee on Analytica Microbiologica, Laboratory technician: person not yet decided
Contractual links to other participants: No.
Objectives: To test the performance and practicability of the developed PCR methods and kit
prototypes compared to traditional methods on routine beer samples and unfiltered process samples in
the Ringnes brewery. To administrate the work in the EBC subcommittee on Analytica Microbiologica
regarding the incorporation of the PCR based analytical standard methods.
Workplan: WP 5, Task 5.3 (8 months): The developed PCR methods/kit prototypes will be tested in
the brewery laboratory. Both test results and practicability as well as running costs will be compared to
routine methods (membrane filtration and incubation on selective media or enrichment in liquid
selective media).
Deliverables: See Table 3.
Partner 7: Brauerei Veltins, An der Streue, D-59872 Meschede-Grevenstein
Scientific team: Stefan Loch-Ahring (Team leader), Hans-Joachim Jahn (Engineer/Scientist)
Contractual links to other participants: No.
Objectives: Veltins Brewery will participate both in the R & D part and the Demonstration part of the
project. As a high value is set on the routine microbiology control, a broad range of samples from the
production will be available for analysis by PCR. The research work will focus on the transfer of the
on-line PCR methods to the routine control of filtered and unfiltered samples. In the Demonstration
part, the practicability of the PCR methods will be examined in a brewery laboratory, and the results
from PCR will be compared with the data from the routine microbiology methods.
Workplan: WP 3 (12 months). Task 3.1 (6). The on-line PCR methods developed by
research partners 2 and 4 will be used to examine real production samples. By continuously
monitoring several sectors of the routine QC microbiology control in parallel with in-house
enrichment methods and PCR, the applicability and sensitivity of the on line PCR methods will
be tested. A special focus will lie on the examination of filtered samples. The different sample
treatment steps like sample filtration, cultivation enrichment etc. up to the isolation of DNA will
be adapted and - if necessary- optimised for use in a routine laboratory.
25
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Task 3.3 (6). The applicability and sensitivity of standard PCR and polyacrylamide gel
electrophoresis will be compared to the on-line PCR method. Both artificially contaminated
samples as well as samples from the routine QC will be processed for PCR and then
analysed in parallel with both PCR systems. The technical performance and the precision of
the methods will be compared. An evaluation of both systems will be given in respect to
common usability in the brewery's laboratory.
WP 5 (8 months). Task 5.3 (8). According to the workplan set in the R & D part, two
demonstration phases will be carried out. Sets of artificially contaminated samples and a
given amount of routine samples from the brewery will be analysed in parallel with PCR
methods and the standard QC protocols. The benefits of PCR compared to microbiology
methods will be assessed. Both the technical side and the possible economical profits will be
taken into consideration.
Deliverables: See Table 3.
Partner 8: Brauerei Beck & Co., Qualitätswesen, P.O Box 107307, Am Deich 18/19, D-28199,
Germany
Scientific team: Dr. Stefan Lustig (Team Leader), Dr. Andreas Eidtman (Scientist)
Contractual links to other participants: No.
Objectives: To develop and implement a fluorometric on line system for the detection of PCR products
in a QA laboratory of an international brewery. To carry out comparative studies of 1) PCR on-line
detection and reference methods (PCR-gel electrophoresis or PCR-ELISA) and 2) PCR methods and
classical microbiological detection media.
Workplan: WP3 (6 months). Task 3.1 (6). Study material in parenthesis.
 Installation, set up and commissioning of a LightCycler (Roche) or TaqMan System (PE Applied
Biosystems) in the brewery.
 Implementation of a fluorogenic on line PCR detection assay (Hyb probe or TaqMan) according to
progress in WP1/2 and the results of preliminary investigations of partners 2 and 4 in WP3.
 Determination of the specificity of the fluorogenic on line detection method using primer sets
developed by partners 1, 2 and 4.
 Determination of the sensitivity of the on-line detection methods using dilution series of test strains.
 Evaluation of possible effects on PCR detection regarding (test strains):
- disinfectant residues in water samples after CIP-procedures
- influence of background flora in water and in beer samples
- influence of trub particles in wort or in unfiltered samples
 Testing of primer cocktails and determination of detection limits (test strains)
 Evaluation and verification of enrichment steps (contaminated beer samples)
 Integration and adoption to workflow in a QA department of an international brewery
(contaminated beer samples)
 Screening of unfiltered, bright and packaged beer samples (contaminated beer samples)
 Screening of contaminated process and rinse water samples (contaminated water samples)
 Method validation
Task 3.3 (6). Study material in parenthesis.
 Comparative analysis of on line PCR with standard PCR methods (contaminated beer samples and
test strains)
 Optimisation and adoption of PCR methods (test strains and contaminated beer samples)
 Comparative analysis of on line PCR detection of process and rinse water samples with ATPbioluminescence (test strains and contaminated beer samples)
WP5 (8 months), Task 5.3.
 Comparative analysis of PCR methods with classical microbiological detection media (e.g. wort
agar, NBB, BSNB, MBH) using test strains and real QA samples from process.
 Validation of method in routine laboratory
Deliverables: See Table 3.
26
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Partner 9: Sternquell Brauerei GmbH Plauen, Dobenaustr. 83, 08523 Plauen, Germany
Scientific team: Dipl. Ing. Thomas Sommer (Team leader), Dipl. Biol. Jürgen Stolle (Scientist)
Contractual links to other participants: No.
Objectives: The role of the partner is to evaluate the developed PCR methods and kit prototypes in a
brewery QC laboratory at Sternquell Brauerei. The partner will provide information about the
suitability of PCR technology to QC environment, about the practicability and performance of the
tested PCR and about the benefits of PCR methods (practical and financial) in comparison with routine
methods.
Workplan: W5 (12 months), Task 5.3. Bright beer samples will be analysed in parallel using the PCR
methods and cultivation on NBB-agar (4 tests/week, 8 samples/test). Unfiltered production samples
will be analysed in parallel with the PCR methods and cultivation in NBB-C (4 test/week, 5
samples/test). The results on both methods will be compared. Considerations include speed,
correspondence of the test results, ease of application and adoption, work safety issues, sensitivity to
DNA contaminations and costs of operation.
Deliverables: See Table 3.
Partner 10: Oxford Brookes University, School of Biological and Molecular Sciences, Headington,
Oxford, OX3 0BP, UK.
Scientific team: Microbiological Team leader: Dr Katherine Smart, Molecular Team Leader: Professor
Linda King, Scientist: to be appointed
Contractual links to other participants: Subcontractor 10, National Collection of Marine and
Industrial Bacteria
Objectives: Co-ordinator of WP4. As an independent expert laboratory, the partner's key role is to
execute objective laboratory based comparison of the developed PCR methodologies and cultivation
based reference methods. In addition, partner will assist in planning, interpretation and reporting of the
method comparison study. Partner will also provide control and test samples for WP1 and WP5.
Dissemination activities include development and execution of training workshops and publication,
reporting and presentation of project outputs.
Workplan: WP1 (2 months), Task 1.1. P10 will prepare artificially contaminated beer samples for the
comparison of the existing PCR assays by P3. Beers (a minimum of 3 types) will be contaminated with
pure cultures of different strains of Lactobacillus, Pectinatus and Megasphaera spp. at concentrations
of 108 to approximately 10 cells per 500 ml beer. The samples will be cold-transported by courier to
P3.
WP4 (21 months), Task 4.1 (2), Task 4.3 (15), Task 4.4 (4). P10 will assist the other partners involved
in the development of the experimental plan. SC10 will provide freeze dried samples for the method
comparison. The samples will be rehydrated in wort, in beer or in unfiltered process samples to
generate “artificial” samples. The team will conduct a comparative analysis of the PCR methods and kit
prototypes developed during the R & D phase of the project and traditional techniques (EBC Analytica
Microbiologica or MEBAK). The team will also assist in the statistical analysis and interpretation of
the data. The findings of WP4 will be reported to WP5 and WP6 in collaboration with the statistician
(SC1).
WP 5 (5 months), Task 5.1 (1), 5.3 (3), 5.4 (1). P10 will assist the other partners to plan and organise
the brewery study by participating in the development of a training strategy and protocol manual to
enable brewery personnel to utilise the PCR methods and kit prototypes. P10 will provide the breweries
with negative and positive controls each month in freeze dried form. The latter comprises mixed and
single populations of microbial contaminants for on site resuspension into beer and process samples to
ensure the assays function correctly.
WP 6 (10 months), Task 6.1 (3), 6.2 (2), 6.3 (5). P10 will assist in the preparation and delivery of
standard methods in IOB Methods of Analysis and in reporting and publishing of the results. The team
will assist in the organisation and execution of two training workshops for end-users to distribute the
theoretical and practical skills needed for implementation of the PCR technology into industrial QC
laboratories.
27
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Deliverables: See Table 3.
Subcontractor 10: National Collection of Marine and Industrial Bacteria (NCIMB), 23 St. Machar
Drive, Aberdeen, AB24 3RY.
Scientific team: Terry Dando, Scientist
Contractual links to other participants: Subcontractor to partner 10, Oxford Brookes University,
School of Biological and Molecular Sciences
Objectives: The main task of the partner is to prepare defined reference material for the objective
comparison of the developed PCR based methods and kit prototypes against the reference methods. The
partner is also responsible for depositing the test organism set to ECCO culture collection.
Workplan: WP4 (4 months), Task 4.2. SC10 will prepare pure and mixed populations of brewery
contaminants in freeze dried format at concentrations in a range designed to examine the limits of the
PCR methods. Fresh, reliably identified brewery isolates representative of Pectinatus, Lactobacillus,
Megasphaera, acetic acid bacteria, wild yeasts and others will be used as test organisms, and they will
be collected among the partners. It is the responsibility of SC10 to place the cultures in the ECCO
culture collection. The reference material will be submitted to P10 as both “known” and “blind”
samples to ensure that objective analysis of the methodologies is achieved. Guidelines for
reconstitution in laboratory media, in unfiltered process samples and in beer will be provided.
Deliverables: See Table 3.
4.
PROJECT MANAGEMENT AND CO-ORDINATION
The outline of the project management is described in Table 4. Four types of groups will be formed
within the project: 1) a Project Management Group (PM), 2) an Industry Platform (IPF), 3) a R&D
Working Group (R&D WG) and 4) a Demonstration Working Group (DEMO WG). The project will be
managed by PM Group consisting of the responsible scientists and industrial representatives assigned
by each contractor and chaired by the co-ordinator. PM Group will keep in regular communication
throughout the project and is responsible for all decisions affecting more than one partner.
Table 4. Project management group.
EUROPEAN COMMISSION
PROJECT
CO-ORDINATOR
PROJECT MANAGEMENT GROUP
Chaired by co-ordinator
- Commission representative
- One industrial representative from each Brewery/Company
- One responsible scientist from each Research Center and University
R&D WORKING GROUP
Chaired by one WP leader
WP 1
WP1
WP
Leader
WP
Partner No 1
Leader
Partner
1
DEMONSTRATION WORKING GROUP
Chaired by one WP leader
WP 2
WP Leader
WP 3
WP Leader
WP 4
WP Leader
WP 5
WP Leader
WP 6
WP Leader
Partner No 3
Partner No 4
Partner No 10
Partner No 2
Partner No 5
INDUSTRIAL PLATFORM
Chaired by one brewery partner
- Brewery partners
- Chairman of EBC Analysis Committee
- Quality control managers of breweries
- Responsible R & D working group leader
- Responsible DEMO working group leader
- SME representative
28
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Project co-ordinator: The project co-ordinator will perform the following functions: oversee the
achievements of the technical objectives of the project in collaboration with the project R&D WG and
DEMO WG ; liase with each of project partners; communicate and interact with the Commission;
supervise preparation and approval of interim and final reports, projects accounts, and other documents
prior to submission to the Commission; chair management meetings; and arrange the final workshop.
Annual reports will be prepared by the co-ordinator and be based on the research and demonstration
results and conclusion collected by R&D and DEMO WG group leaders from workpackage leaders.
The agenda of the project management meetings will be set by the project co-ordinator. The
responsible EC scientific officer will be informed about scheduled meetings and their agenda at least
eight weeks in advance by the co-ordinator. The co-ordinator will also take the minutes, note the
actions, and circulate these to the project partners within two weeks following the meeting. Issues that
will be covered are: Reports from working groups; deliverables in line with workpackage
commitments; modifying workplans for the following phase of the project based on the achievements
to date; punctuality of reporting; financial issues (guidelines on completion of cost statements);
intellectual property issues for maximising the economic and technological impact of the project;
dissemination issues; discussion of other matters which may have arisen since the previous meeting.
Industrial platform: IPF consist of brewery participants, chairman of European Brewery Convention
(EBC) Analysis Committee, quality control managers of breweries involved in the project, a SME
representative and responsible WG leaders. In this platform the responsible WG leaders communicate
all information relating to the process of workpackages to the brewery participants. This platform will
ensure critical evaluation and standardisation of novel methodologies developed and demonstrated.
R&D and demonstration working groups: R&D and DEMO WGs are formed around workpackages
and consist of scientific teams and industrial representatives of the consortium (partnership). Both
groups are lead by one workpackage leader responsible for the execution of all workpackages and for
reporting the out-come to the project co-ordinator. These groups will deal with exchange of information
and material, monitor the scientific and demonstration progress, compare achievements with milestones
set out in the proposal, prepare annual progress reports and publications etc.
Meetings: The time schedule and frequencies of the meetings of established groups throughout the
project is presented in Table 5. PM meetings are held at the outset of the project and at yearly intervals.
Beside PM meetings the IPF meetings will be held on a yearly basis in conjunction with the
management meetings. In addition R&D and DEMO WG meetings will be scheduled at intervals of six
months on the rotating basis at different host laboratories and breweries. A workshop will be arranged
in the end of the project. It will be open in addition to participants of all 21 member countries of EBC
also to participants of non-EBC countries. Symposia and training workshops will be organised to
ensure full exploitation and adoption of the technology by the European brewing industry.
Table 5. Time schedule of meetings.
1. year
6
2. year
Month 1
PM
R&D WG
DEMO WG
IPF
12
PM
R&D WG
DEMO WG
IPF
R&D WG
PM;
R&D WG;
DEMO WG;
IPF;
Project Management Group
R&D Working Group
Demo Working Group
Industrial Platform
18
R&D WG
DEMO WG
29
24
PM
R&D WG
DEMO WG
IPF
3. year
30
R&D WG
DEMO WG
36
PM
R&D WG
DEMO WG
IPF
WORKSHOP
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
Decision-making structures: The co-ordinator has the ultimate responsibility for decision made within
the project. For major decisions, a consensus approach will be used at the PM meetings. Minor issues
that require decisions will be addressed via e-mail through co-ordinator to the other partners and
options taken by return e-mail. Serious issues should be addressed at PM meetings. If the situation
requires urgent attention, a special PM meeting will be convened. In the unlikely event of a conflict
between partners, it will be role of the co-ordinator to act as mediator and resolve the conflict.
Quality assurance and monitoring of project progress: The assessment criteria for progress monitoring
will include: 1) Deliverables in line with the workpackages, 2) Internal control results from the working
groups, 3) Adherence to best practice and conformity with documented procedures, 4) Feed back from
end-users through Industry Platform, 5) Financial revisions. Internal control for correct identification of
milestones and achievements of results will be done through the periodic PM meetings. Responsible
scientists representing each individual contractor are accountable for the quality of work carried out in
their own institutes or companies. The demonstration work carried out in industry will be overseen
locally by brewing quality assurance management and monitored by central office technical staff of the
research partners.
Intellectual properties and legal issues: The back ground information each partner brings to the
BREWPROC project will be documented and agreed by the other partners and will be deposit with the
project co-ordinator. Each partner will be working with their individual patent advisor to identify
possible patenting opportunities and to try clarify their rights and ability to exploit fore ground
information generated in the project. A separate Consortium Agreement will be signed defining the
necessary rules for management of the project, user rights and exploitation of results as well as liability
questions. Intellectual property rights are applied either by the participant having generated the results,
or under a separate agreement by the industrial participant aiming at commercialisation of the same.
Total person-months co-ordination: 11
5.
EXPLOITATION AND DISSEMINATION ACTIVITIES
Exploitation plan
The user groups of the knowledge and technological outputs of the project, i.e. beverage producing
companies and companies supplying materials and kits for exploiting the technology to end users, are
targeted by the dissemination and exploitation plan.
Exploitation of the developed technology: This project will evaluate a number of approaches to a rapid
and sensitive technology for detection of contaminants possibly occurring during the production and in
the products of the brewing industry. This will result in development of specific detection methods and
the subsequent commercialisation of analytical kits produced by the SME partner PIKA Weihenstephan
GmbH according to a separate agreement by the partners.
The deliverables from R&D part will be disseminated directly to PIKA Weihenstephan GmbH to be
used for the production of Test Kit prototypes. These prototypes will then be passed to the participating
breweries who will test them in their QC laboratories on samples from their own routine control. These
steps will lead to a direct transfer of methods and knowledge not only from the research partners,
Universities and Research Institutes, to industry (SME and beverage producers), but also the other way
back and give valuable information about the actual needs form industry to the research groups.
Agreements between partners: A consortium agreement will be signed between the partners to lay
down the rules for exploitation. Every partner will receive the rights for in-house use of the methods;
PIKA Weihenstephan GmbH will in addition get the rights concerning the production of kits resulting
from the developments in the project. The rights on the different development steps done by different
30
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
partners and possibly leading to a commercialised product will be covered by an intellectual property
plan which will be prepared in advance of the practical works.
In accordance to a separate agreement between the partners the assays or parts of them developed
during this project are planned to be patented with the partner(s) of the project doing the work that lead
to those results as inventors.
Licensing will be carried out in a manner that enables a high cumulative volume in consumer markets.
After finishing the project, external organisations will get the possibility to buy licenses to use the
developed methods. Other breweries than the partners of the project will get license rights for in-house
use of the developed analysis methods, and further licenses will be given to non-partners, preferably
SME's, throughout the EU to use the kits and PCR methods for customer's supply.
Commercialisation plan: The participating SME will involve the newly developed products into its
business plan and put high efforts in merchandising the products in all fields of the food processing
industry. As there are already close negotiations with different companies producing the instruments
needed for the PCR analyses and who also have a high interest to developing the food market, PIKA
Weihenstephan has a perfect base for commercialisation of the developed products.
The marketing strategies for the new technology will aim first on breweries, but soon also on users in
other fields, starting with the beverage industry in general and then ongoing to other fields. Workshops
will be held in the laboratories of the SME on a routine basis to give future customers the possibility to
test the methods with real samples and get familiar with their use and the resulting possible benefits for
their companies.
Obstacles for exploitation: Perceived obstacles for the exploitation are mainly the technical
performances of the methods. The possibilities for the development of a sensitive and reliable test
system are very good, but all PCR methods developed so far originate from specialised research
laboratories. First results from a pilot study carried out by Dr. Vogeser D-TECT (from which PIKA
Weihenstephan is the legal successor) together with some breweries showed already the possible
benefits of the new methods compared to the old ones nowadays used, but also problems with the
applications itself occurred. A major risk for the implementation of the new methods into the QC
laboratories therefore will be the adaptation of the developed methods to the laboratory routine, both
concerning their handling and their possible implementation in the daily routine of a food production
control laboratory.
Dissemination plan
Dissemination to the European brewing industry: The existing network of the European Brewery
Convention (EBC ) with its Committees and Groups as well as its Congresses and Symposia will be
used as platforms for dissemination of the deliverables of the project. The EBC Detection of
Contaminants Subgroup and the EBC Brewing Science Group will be utilised for changing and
dissemination of ideas and achievements during the project. The meeting of the Detection of
Contaminants Subgroup at mid term of the project will be arrange for reporting project results. At the
next full Congress of the EBC near the project end in Dublin in 2003, a full Congress Session will be
devoted to the results and, most importantly, adoption experiences of the output of the project by the
industry after completion of the project. A separate Symposium devoted to communicating the outputs
of the project will be organised during the first year following the end of the project. This Symposium
will be directed to an extended audience including wine, cider and other beverage producing industry
as well as authorities in addition to brewing industry.
The EBC website (www.ebc-nl.com) will be used for distribution of information concerning the
ongoing project. Web pages for inclusion in or linkage to the web sites of production companies,
research institutes, EU and European Brewing Convention will be constructed. These will give on line
31
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
help, a contacts database, a reference of scientific publications, and a database of those trained in the
technology.
The methods will finally be made available to European breweries by incorporating them in the the
manuals EBC Analytica Microbiologica, IOB Methods of Analysis and MEBAK (Mitteleuropäische
Brautechnische Analysenkommision).
Reports, articles and publications: The partners of the project will produce reports on the key results.
Initially these will be distributed to the members of the project, but when appropriate, later published in
relevant scientific and technical journals thereby disseminating the results of the project to other food
and beverage sectors and to customer groups. Academic and research partners will write scientific
papers according to their individual contributions and results. Joint or sole authorship will be resolved
according to the usual conventions of degree of practical and intellectual input to any element of
publishable material. The actions under WP6 regarding scientific publications will be to ensure that any
information that has not been captured by publication is written for submission to appropriate journals.
The task of the working group will also be to prepare a reference list of publications arising from the
project for separate publication, including the World Wide Web. The decision to produce a monograph
containing both scientific and applied technical information will be made at the WP6 stage.
Training Workshops: Training workshops will be arranged to ensure full exploitation and adoption of
the technology by the European brewing industry. Workshops will cover theory and provide
opportunity for practical work. To ensure ease of access by all regions of the EU, they will be held at a
number of partner institutes with appropriate teaching and laboratory facilities. Due to the low numbers
of delegates which can be accommodated on such intensive courses, a programme of repeat courses
during the first 2 years after the end of project will be an output of WP6.
6.
ETHICAL ASPECTS AND SAFETY PROVISIONS
There are no potential negative impacts of a social, environmental or economic nature likely to stem
from this project proposal (QLRT-1999-31251). The proposal does not involve research on persons,
genetic modification of animals or plants, the use of human embryos or foetus, human embryonic or
foetal tissue, other human tissue, non-human primates or other animals.
All proposed experimental work will be carried out in accordance with relevant EU and International
Safety and Environmental legislation. Micro-organisms used in the experimental work are all nonpathogenic belonging to Risk Group 1 and considered as "Non-infectious Perishable Biological
Substances" (NPBS). Detailed regulation for the packaging and transport of NPBS published by
Universal Postal Union (UPU Convention 1984) will be followed.
32
BREWPROC
Technical annex
Project proposal Nr QLRT-1999-31251
ANNEX 1. Time table of project activities by task.
YEAR 1
WPs &
tasks
1
WP1
Task 1.1
Task 1.2
Task 1.3
Task 1.4
Task 1.5
WP2
Task 2.1
Task 2.2
Task 2.3
Task 2.4
Task 2.5
WP3
Task 3.1
Task 3.2
Task 3.3
WP4
Task 4.1
Task 4.2
Task 4.3
Task 4.4
WP5
Task 5.1
Task 5.2
Task 5.3
Task 5.4
WP6
Task 6.1
Task 6.2
Task 6.3
Meetings 
SC=subcontractor
3
5
7
9
11
13
YEAR 2
Months
17
19
21
4
15
1
YEAR 3
23
25
27
29
31
33
35
36-42
^
^
^
^
^
^
^
^
2
^
5
6
^
^
^
^
^
^
^
^
^
^
8
^
^
^
7
^
^
^
^
^
^
9
^
^
^
3
^


^
^
^

^

33
^
10
^
^


11
^
Partners
1 (R)
3, 10
1, 2, 4
1, 2, 4
1, 2
4
3 (R)
1, 2, 4
1, 3
1, 2, 4
1, 2
4
4 (R)
2, 4, 7, 8
3
2, 3 ,7, 8
10 (R)
1, SC1, 10
SC10
10
SC1, 10
2 (R)
1-4, SC1
1-4
1-10
1-4, SC1
5 (R)
1-5, 10
1-5, 10
2, 4, 10