Assessing barley malt associated microbial diversity using next generation sequencing

Transcription

1 Assessing barley malt associated microbial diversity using next generation sequencing Mandeep Kaur 1, Evan Evans 1, Doug Stewart 2, Agnieszka Janusz 2, Barbara Holland 1 and John Bowman 1 1 University of Tasmania, Hobart, Australia 2 Viterra Ltd, Adelaide, Australia

2 Introduction

3 Microbial contamination of grains During storage and transport of barley During Malting In the field

4 Microbes, barley, malt and malting a dynamic ecosystem traditional understanding 1.E+08 1.E+07 Viable counts 1.E+06 1.E+05 1.E+04 Aerobic heterotrophic bacteria Lactobacilli Moulds yeasts 1.E+03 1.E+02 Stored barley First steep Second steep Green malt Stage of malting Kilned malt Screened malt (extracted from Petters et al 1988)

5 Why study barley malt associated microbiota? (Laitila 2007)

6 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 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 Toxins DON, OTA, aflatoxins, zearaleneone Allergens posing human health risks Qualitative and quantitative changes in cereal components 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)

7 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).

8 Cloning and sequencing Genomic DNA extraction PCR LSU rrna gene with fungal specific primers Ligate LSU rrna gene into plasmids Transform E coli cells with plasmids PCR with sequencing primers and run on automated sequencer Grow clones on plates

9 Tag encoded GS FLX 454 amplicon pyrosequencing

10 Comparison of pyrosequencing results for eight malt samples

11 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

12 MEGAN (MEtaGenome ANalyzer) comparison of large subunit (LSU) rrna gene reads collected from all eight different barley malt samples using pyrosequencing

13 MEGAN (MEtaGenome ANalyzer) comparison of large subunit (LSU) rrna gene reads collected from all eight different barley malt samples using pyrosequencing

14 MEGAN analysis of large subunit (LSU) rrna gene reads collected from different barley malt samples

15 Principle component analysis of pyrosequencing data PYF predicted Scores pos pos pos neg neg neg pos Factor-2 (20%, 10%) Factor-1 (34%, 89%) pos

16 Principle component analysis of pyrosequencing data PYF predicted

17 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.

18 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.

19 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