Assessing barley malt associated microbial

diversity using next generation sequencing

Mandeep Kaur1, Evan Evans1, Doug Stewart2, Agnieszka

Janusz2, Barbara Holland1 and John Bowman1

1University of Tasmania, Hobart, Australia

2Viterra Ltd, Adelaide, Australia
Introduction
 Microbial contamination of grains

During storage and

During Malting
transport of barley

In the field
Microbes, barley, malt and malting
• a dynamic ecosystem
• traditional understanding
1.E+08

1.E+07
Aerobic heterotrophic
bacteria
1.E+06 Lactobacilli
Viable counts

Moulds
1.E+05
yeasts

1.E+04

1.E+03

1.E+02
First steep
Stored barley

Kilned malt

Screened malt
Second steep

Green malt

Stage of malting

(extracted from Petters et al 1988)

Why study barley malt associated
microbiota?

(Laitila 2007)
Microbial implications from grass to glass – an overview
Negative effects of microbes
Plant diseases
Inhibition of grain germination
Metabolites causing down the process technical
problems:
• premature yeast flocculation (PYF)
• gushing
• exopolysaccharides reducing wort separation
• organic acids causing variation in wort pH
Toxins – DON, OTA, aflatoxins, zearaleneone
Allergens posing human health risks
Qualitative and quantitative changes in cereal
components
Positive effects of microbes
Source of farm fungicides - bio control or
inhibition of undesirable microbes
Enhancement of grain germination–GA, IAA,
ABA etc
Source of hydrolytic enzymes:
• amylases
• β- glucanases
• proteases
• xylanases
Food grade biocontrol agents – antimicrobial
factors
Health promoting compounds – enzymes,
organic acids, phenolic compounds and
vitamins
Reduction in malting losses
(More good than bad - Laitila 2008)
 Aim of study
• Examine the fungal microbial communities associated
with commercial malts using molecular techniques to
improve brewing uses.

• Avoid potential brewing problems (i.e. PYF, mycotoxins).

• Enhance opportunities for favourable microbial benefits

(e.g. enzymes, hormones, malt pH).
 Cloning and sequencing

Genomic DNA extraction PCR Ligate LSU rRNA gene

LSU rRNA gene into plasmids
with fungal specific primers

Transform E coli cells

with plasmids

PCR
with sequencing primers
and run on automated sequencer Grow clones on plates
Tag encoded GS FLX 454 amplicon pyrosequencing
Comparison of pyrosequencing results for
eight malt samples
 Rarefaction curves illustrating the effect of large
subunit (LSU) rRNA gene partial sequence number on
the number of operational taxonomic units (OTUs)
identified from the eight barley malt samples
 MEGAN (MEtaGenome ANalyzer) comparison of large subunit
(LSU) rRNA gene reads collected from all eight different barley
malt samples using pyrosequencing
 MEGAN (MEtaGenome ANalyzer) comparison of large subunit
(LSU) rRNA gene reads collected from all eight different barley
malt samples using pyrosequencing
MEGAN analysis of large subunit (LSU) rRNA gene reads
collected from different barley malt samples
 Principle component analysis of
pyrosequencing data – PYF predicted
Scores
1.4
pos pos
1.2

0.8 pos
0.6

0.4

0.2

-0.2
neg
neg neg
-0.4

-0.6
pos
-0.8
Factor-2 (20%, 10%)

-1

-1.2
pos
-1.4

-1.6
Factor-1 (34%, 89%)
-2 -1 0 1 2
 Principle component analysis of
pyrosequencing data – PYF predicted
 Future work
• Comprehend the precise role these fungi play in the
multifaceted grain based ecosystem.

• Design specific qPCR primers to select for (e.g. pH

acidification) or avoid microbes (e.g. PYF causing).

• Tailor malting conditions that result in better quality,

safe and sustainable end product.
 Conclusions
• Microbes are an integral part of barley malt
ecosystem.

• Microbial activities greatly influence malt safety and

quality.

• Optimisation of malt production and quality demands

a balance between good and bad microbes.

• New molecular techniques like pyrosequencing

seems to have an edge over traditional cloning and
sequencing in exploring the barley malt microbial
ecology.
 Acknowledgements
Co authors
Drs Evan Evans, Barbara Holland and John Bowman (UTAS)
Drs Doug Stewart and Agnieszka Janusz (Viterra Ltd)

Funding:
Grains Research and Development Corporation
Viterra Ltd