DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria: Dissertation

Research output: ThesisDissertation

1 Citation (Scopus)

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 languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Haikara, Auli, Supervisor, External person
Award date11 Dec 2009
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-7369-1
Electronic ISBNs978-951-38-7370-7
Publication statusPublished - 2009
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Selenomonadales
brewing industry
Megasphaera cerevisiae
beers
Megasphaera
DNA
Zymophilus raffinosivorans
Pectinatus
bacteria
Selenomonas lacticifex
Pectinatus cerevisiiphilus
Pectinatus frisingensis
Selenomonas
methodology
biodiversity
enrichment culture
spoilage bacteria
monitoring
off flavors
assays

Keywords

  • beer
  • brewing
  • identification
  • PCR
  • Pectinatus
  • phylogeny
  • Megasphaera
  • rapid detection
  • Selenomonas
  • spoilage
  • taxonomy
  • Zymophilus

Cite this

@phdthesis{86ee1142300c4af3ae812771c6528278,
title = "DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria: Dissertation",
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.",
keywords = "beer, brewing, identification, PCR, Pectinatus, phylogeny, Megasphaera, rapid detection, Selenomonas, spoilage, taxonomy, Zymophilus",
author = "Riikka Juvonen",
note = "Project code: 34261",
year = "2009",
language = "English",
isbn = "978-951-38-7369-1",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "723",
address = "Finland",
school = "University of Helsinki",

}

DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria : Dissertation. / Juvonen, Riikka.

Espoo : VTT Technical Research Centre of Finland, 2009. 140 p.

Research output: ThesisDissertation

TY - THES

T1 - DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria

T2 - Dissertation

AU - Juvonen, Riikka

N1 - Project code: 34261

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

KW - beer

KW - brewing

KW - identification

KW - PCR

KW - Pectinatus

KW - phylogeny

KW - Megasphaera

KW - rapid detection

KW - Selenomonas

KW - spoilage

KW - taxonomy

KW - Zymophilus

M3 - Dissertation

SN - 978-951-38-7369-1

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -