Microbial community in barley

The microbial community characteristic to malted barley products develops in the field, under storage, and during the processing (Figure 3). Many intrinsic and extrinsic factors including plant variety, climate, soil type, agricultural practices, storage and transport influence the diversity and structure of the microbial community present in the barley grains (Angelino & Bol 1990, Douglas &

Flannigan 1988, Flannigan 2003, Haikara et al. 1977, Petters et al. 1988). Of these climate is believed to play a particularly important role (Etchevers et al.

1977). Therefore, barleys cultivated in different geographic locations have different microbial communities. The composition of the microbial community on barley grains changes dramatically as a result of post-harvest operations (Figure 3). Some of the grain-associated microbes are removed during processing of grains, whereas every process step in the barley-malt-beer chain can be a source of additional microbial populations. A stored barley batch as well as the grain bed in malting can be considered as a man-made ecosystem, in which the live barley tissues can interact with the surrounding environment and microbes.

Figure 3. Three ecological niches for microbial communities in malting barley.

Barley grain is composed of three major parts: the embryo, the endosperm and a protective layer including the husk, the pericarp and the testa, also known as the seed coat (Figure 4). The husk mediates uniform water uptake and provides mechanical protection for the barley embryo and the primary leaf developing during the germination (the acrospire). The several different layers found in the grain coverings act as a carrier for microbes (Olkku et al. 2005). In the field,

Field

barley kernels are already colonized by microbes soon after ear emergence from the enveloping leaf-sheaths. Wind, rain, insects, birds and agricultural practices effectively distribute microbes throughout the growing season (Flannigan 2003).

At later stages of kernel filling, microbial colonization is restricted to the outer parts of the developing kernels, between the testa and the outer epidermis. In healthy grains, testa restricts microbial attack into the grain interior (Figure 4C).

Occasionally, invasion of the endosperm is caused by fungi with distinct phytopathogenic characteristics, such as Fusarium fungi, or if the testa is for some reason injured (Schmidt 1991).

Figure 4. A) Barley grain structure. B) Structure of the outer layers in mature barley grain (reference Olkku et al. 2005). C) Microbial biomass located outside the testa layer.

Barley kernels represent a complex, non-uniform substrate for microbes with respect to physical and chemical parameters (Noots et al. 2003). Barley has the following average chemical composition: total carbohydrate 70–85% (including

aleurone layer

starch, cellulose, β-glucans, pentosans and gums), protein 10.5–11%, inorganic matter 2–4%, fat 1.5–2.0% and other substances 1–2% (including polyphenols, vitamins) (Kunze 1999, Palmer 1989). The majority of the nutritional components are accumulated in the endosperm cells. The outer layers of grains, in which the significant part of the microbial community is located, consist mainly of cellulose, hemicellulose and lignin and also contain small amounts of proteins (Olkku et al. 2005).

It has been suggested that microbial populations adhered to external and internal surfaces of barley tissues form a compact biofilm (Thomas & Usher 2001). This multicellular mode of growth predominates in nature and provides adaptive strategies for plant-associated microbes in changing or stressful environments (Morris & Monier 2003). In a nutrient-poor environment such as on the surfaces of plant tissues, microbial cells often become filamentous to maximize their absorbing surface (Morris & Monier 2003). Biofilm-grown cells are also well protected and have shown increased resistance to external factors such as desiccation, heat and antimicrobial treatments (Costerton et al. 1987). However, little is known about the complex associations of microbes within grain biofilms during barley processing.

The indigenous microbial community of barley harbours a wide range of microorganisms including numerous species of Gram-negative and -positive bacteria, yeasts and filamentous fungi (Flannigan 2003, Haikara et al. 1977, Noots et al. 1999, Petters et al. 1988). Low levels of actinobacteria, mainly members of the Streptomycetes genus, occur occasionally. Table 1 shows microbes frequently detected on pre-harvest barley.

Bacteria numerically dominate the culturable microbial community of pre-harvest barley (Angelino & Bol 1990). Approximately 10 million bacteria are frequently detected in one gram of barley (Flannigan 2003, Noots et al. 1999).

This provides an estimate that at least 500 000 bacteria can be found in a single barley kernel.

Table 1. Microbial species belonging to the listed genera are frequently detected on pre-harvest barley (Flannigan 2003, Haikara et al. 1977, Noots et al. 1999, Petters et al. 1988).

Bacteria Yeasts Filamentous fungi Bacillus Candida Alternaria

Enterobacter Cryptococcus Aureobasidium Erwinia Pichia Cephalosporium Flavobacterium Sporobolomyces Cladosporium

Klebsiella Rhodotorula Dreachslera

Micrococcus Trichosporon Fusarium

Pseudomonas Epicoccum

Streptomyces Xanthomonas

Yeasts are the second most abundant culturable microbes in pre-harvest barley (Flannigan 2003). However, their numbers may be exceeded by filamentous fungi during later stages of ripening (Angelino & Bol 1990, Flannigan 2003).

More than 150 species of filamentous fungi (moulds) and yeasts can be found on grains as surface contaminants or as internal invaders (Sauer et al. 1992).

Filamentous fungi are divided into two distinct ecological groups: field and storage fungi. Among the most common and widespread field fungi in malting barley are Alternaria, Cladosporium, Epicoccum, Fusarium, Cochliobolus, Drechslera and Pyrenophora, the latter three formerly known as Helminthosporium-group (Ackermann 1998, Andersen et al. 1996, Flannigan 2003, Haikara et al. 1977, Noots et al. 1999). These fungi require relatively high water availability for growth (a_w > 0.85). Thus, their growth is restricted during storage by appropriate drying of barley.

After harvest, barley grains are stored from about two months to one year to allow the break up of the normal dormancy before malting (Pyler & Thomas 2000). Microbes are not usually active and their number generally decreases during storage under appropriate conditions (Beck et al. 1991, Haikara et al.

1977, Laitila et al. 2003). Microbial growth and spoilage of stored barley are determined especially by water activity and temperature (Angelino & Bol 1990).

Xerophilic Aspergillus, Eurotium and Penicillium are the most characteristic fungi found in the storage environment (Pitt & Hocking 1997, Samson et al.

2000). Storage fungi are habitually present in the dust and air of the storage environment, and can also be found in different farm and malting equipments such as harvesters and elevators (Sauer et al. 1992). However, the differentiation into field and storage fungi is applicable only in temperate climates, since in warmer regions some species normally considered as storage fungi may be found already in the developing barley (Medina et al. 2006, Noots et al. 1999).