Abstract
Megasphaera cerevisiae, Pectinatus cerevisiiphilus,
Pectinatus frisingensis, Selenomonas lacticifex,
Zymophilus paucivorans and Zymophilus raffinosivorans are
strictly anaerobic Gram-stain-negative bacteria that are
able to spoil beer by producing off-flavours and
turbidity. They have only been isolated from the beer
production chain. The species are phylogenetically
affiliated to the Sporomusa sub-branch in the class
"Clostridia". Routine cultivation methods for detection
of strictly anaerobic bacteria in breweries are
time-consuming and do not allow species identification.
The main aim of this study was to utilise DNA-based
techniques in order to improve detection and
identification of the Sporomusa sub-branch beer-spoilage
bacteria and to increase understanding of their
biodiversity, evolution and natural sources. Practical
PCR-based assays were developed for monitoring of M.
cerevisiae, Pectinatus spp. and the group of Sporomusa
sub-branch beer spoilers in brewery process and product
samples. The developed assays reliably differentiated the
target bacteria from other brewery-related microbes. The
contaminant detection in process samples (101-103 cfu
ml-1) could be accomplished in 2-8 h. Low levels of
viable cells in finished beer (?10 cfu 100 ml-1) were
detected after 1-4 d culture enrichment. Time saving
compared to cultivation methods was up to 6 d. Based on a
polyphasic approach, this study also revealed the
existence of three new anaerobic spoilage species in the
beer production chain, i.e. Megasphaera paucivorans,
Megasphaera sueciensis and Pectinatus haikarae. The
description of these species enabled establishment of
phenotypic and DNA-based methods for their detection and
identification. The 16S rRNA gene based phylogenetic
analysis of the Sporomusa sub-branch showed that the
genus Selenomonas originates from several ancestors and
will require reclassification. Moreover, Z. paucivorans
and Z. raffinosivorans were found to be in fact members
of the genus Propionispira. This relationship implies
that they were carried to breweries along with plant
material. The brewery-related Megasphaera species formed
a distinct sub-group that did not include any sequences
from other sources, suggesting that M. cerevisiae, M.
paucivorans and M. sueciensis may be uniquely adapted to
the brewery ecosystem. M. cerevisiae was also shown to
exhibit remarkable resistance against many
brewery-related stress conditions. This may partly
explain why it is a brewery contaminant. This study
showed that DNA-based techniques provide useful tools for
obtaining more rapid and specific information about the
presence and identity of the strictly anaerobic spoilage
bacteria in the beer production chain than is possible
using cultivation methods. This should ensure financial
benefits to the industry and better product quality to
customers. In addition, DNA-based analyses provided new
insight into the biodiversity as well as natural sources
and relations of the Sporomusa sub-branch bacteria. The
data can be exploited for taxonomic classification of
these bacteria and for surveillance and control of
contaminations.
Original language | English |
---|---|
Qualification | Doctor Degree |
Awarding Institution |
|
Supervisors/Advisors |
|
Award date | 11 Dec 2009 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 978-951-38-7369-1 |
Electronic ISBNs | 978-951-38-7370-7 |
Publication status | Published - 2009 |
MoE publication type | G5 Doctoral dissertation (article) |
Keywords
- beer
- brewing
- identification
- PCR
- Pectinatus
- phylogeny
- Megasphaera
- rapid detection
- Selenomonas
- spoilage
- taxonomy
- Zymophilus