Assuring the microbial safety and quality of Australian malt and barley

Australia produces approximately 32% of the world's traded malting barley, ranking number one in the world for malting barley export. In the international export market, Australia has traditionally had a reputation for producing clean and bright barley/malt, which is presumably due to low microbial loads, resulting from dry conditions occurring during maturation and harvest. The diverse microbial communities naturally colonizing barley grains greatly influence malt quality, and subsequently other products in the malt value chain, in particular beer. The objective of this thesis is to comprehend microbial diversity associated with barley and malt, thereby leading to a better understanding of cause of factors (such as microorganisms) that produce mycotoxins and cause premature yeast flocculation which impact brewing efficiency and beer quality. Microbial fingerprinting of Australian malt and barley grown in different regions was benchmarked against malting barley grown internationally by using terminal restriction fragment length polymorphism {TRFLP) analysis. This approach was supported by cloning and sequencing techniques to assess microbial population composition. The TRFLP approach was considered the most appropriate because it is comparatively rapid, cost efficient and that microbial profiles from a large number of samples can be assessed. The TRFLP approach uses amplification of generic primers for the bacterial 16S rRNA gene and Dl/D2 domain of the fungal 26/28S rRNA gene. Both qualitative and quantitative differences were observed in bacterial and fungal communities associated with malts produced in different geographical regions. The TRFLP and cloning approaches identified a greater diversity in yeast and filamentous fungi associated with barley malts than previously reported. Presumably this is the result of TRFLP being a culture independent approach, compared to traditional \wet plate\" culture techniques which can bias towards the selective enrichment of fast growing microorganisms adapted to high substrate concentrations that can potentially represent a minor fraction of the resident microbial community. Considerable differences in terms of bacterial and fungal populations were observed between Australian barley samples and their corresponding malts. The malts produced in different malt houses were dissimilar in terms of fungal community structure. Fungal clone libraries of different barley and malt samples demonstrated the absence of Fusarium graminearum Aspergillus and Penicillium spp. the sources of deoxynivalenol (DON) and ochratoxin A (OTA). The absence of these mycotoxins in Australian malt was verified by testing malt samples for DON and OTA. A new procedure for the regeneration of DON and OTA immunoaffinity columns was developed and used for detecting these mycotoxins in Australian barley and malts. This new regeneration method reduces the cost of screening for these commonly tested mycotoxins. None of the samples were found to contain detectable levels of either of DON or OTA. This outcome was attributed to the typically dry to hot climatic conditions in the Australian barley growing regions during the period from heading to maturity of the barley crop. In addition temperatures below 20°C at anthesis avoid the optimal conditions required for the infection of DON-producing Fusarium graminearum strains. Furthermore the dry harvest conditions result in dry barley with moisture content (in this study average 10.9% with a range of 8.7 - 12.4%) <13% for storage which is well below the minimum moisture content (>14%) that is conducive for the growth of OTA-producing Aspergillus and Penicillium spp. Premature yeast flocculation (PYF) is an intermittent brewing fermentation problem that results in incomplete wort fermentation and is a significant problem for some breweries. The traditional approach to avoiding and solving this problem has been to detect PYF positive malts by using a small scale fermentation test. These fermentation tests are time consuming expensive sometimes inconsistent and difficult to transfer between testing laboratories. Research has also been directed at identifying the causal wort components (pectin/arabinoxylan or protein) of PYF. Neither of these approaches has been particularly successful over the past 40+ years. Consequently the problem was approached from a different and novel perspective. That was to use molecular finger printing as a step to identify the microbial taxa that cause PYF by comparing positive and negative malts using TRFLP cloning and sequencing. A significant breakthrough has been made with this approach and a concept developed identifying substantial differences between PYF positive and negative malts in their TRFLP (Haelll digestion) fungal fingerprints using the generic primers for the Dl/D2 domain of the fungal 26/28S rRNA gene. This analysis indicates that more than one taxon of fungi are associated with PYF which perhaps indicates why previous researchers have had difficulty identifying the causal microbial taxa and causal agent/s."