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The microbial community of brewing barley and malt

In general, the microbial community found on or in barley seeds may contain numerous species from five principle groups, i.e. viruses, bacteria, fungi, slime moulds and protozoa. The presence ofviruses on the surface of barley seeds or within their interior parts is a generally unexplored field of research. Because of its role as a seed borne pathogen of barley, the barley stripe mosaic virus and its association with barley grains have been studied more intensively (Mink, 1993). However, the presence and impact of symptomless barley viruses on the microbiota present on or in the barley seed has not been studied to date. Also the impact that viruses associated with fungi (mycoviruses) or bacteria (bacteriophages) may have on individual species or on the community of microorganisms present on or in barley seeds is as yet unknown. However, an influence can be assumed since many plant pathogenic fungi and endophytic fungi of grasses have been found to carry mycoviruses (see comprehensive literature compilations by Pearson et al. (2009) and by Herrero et al. (2009). According to the literature, many fungal species undergo modifications of their phenotype leading to increased or decreased virulence of virus infected strains of plant pathogens. This effect has been observed in species which are important to the quality and yield of barley, e.g. F. graminearum or F. culmorum (Chu et al., 2002), Alternaria alternata (Aoki et al., 2009) including potential production of mycotoxins by Aspergillus species (Varga et al., 1994). On the other hand, the link between mycoviruses and the production of fungal secondary metabolites has been observed as well (Detroy and Worden, 1979). Where knowledge about specific interaction of fungal viruses with their host and of the impact of this interaction on the barley microbiota is scarce, knowledge of this relation in bacteria and their respective phages is even more negligible. However, this relation has been intensively studied in areas such as medicine (Merril et al., 2003), biotechnology or food fermentation, where bacteriophages play an important role (Emond and Moineau, 2007). It can therefore be speculated that bacteria present on or in barley seeds will interact with bacterial viruses and that their numbers and activity will also be influenced, either positively or negatively.

Occurrence of slime moulds (Mycetozoa) and protozoans on cereal seeds has been reported by some authors (Pepper and Kiesling, 1963; Mills and Frydman, 1980). However, no representative data about their presence or about the species and groups (plasmodial or cellular slime moulds, amoeboid or ciliate protozoa) prevailing have been presented or published elsewhere. The fact that slime moulds and protozoa feed on living and dead bacteria, yeasts, and fungi might point to a certain yet unspecified effect of slime moulds and protozoans on the microbial community when present on barley seeds (Hohl and Raper, 1993).

Bacteria together with fungal organisms can be supposed to be the groups with the greatest influence on the properties of barley grain since they occur regularly in higher numbers and many of them are physiologically able to use grain components as nutrient source. According to Roberts et al. (2005), the bacterial consortium consists of aerobic mesophilic bacteria (no growth at 10°C or less, optimum growth between 20°C and 40°C, colony count from 102 to 106 cfu/g), psychro- trophic bacteria (growth occurs at 7°C or less, growth optimum > 20°C, colony count from 104 to > 105 cfu/g), actinomycetes (up to 106 cfu/g), aerobic spore-forming bacteria (colony counts from 100 to 105 cfu/g) and coliform bacteria (colony counts from 102 to 104 cfu/g). An overview of bacterial species that have been isolated from barley grain was compiled by Pepper and Kiesling (1963) and, more recently, by Noots et al. (1999). The spectrum shows a variety of different species. Many of them belong to Gram-negative genera such as Alcaligenes, Clavibacterium, Enterobacter, Erwinia, Escherichia, Flavibacterium, Pseudomonas or Xanthomonas. Gram positive species represent the genera Arthrobacter, Bacillus, Corynebacterium, Lactobacillus, Leuconostoc, Micrococcus and Ther- moactinomyces. Some genera are represented by only one species, e.g. Arthrobacter, Aplanobacter, Brevibacterium, Corynebacterium, Kurthia and Pediococcus. Moreover, groups of unidentified bacteria were summarized as white and yellow bacteria. Filamentous bacteria were summarized as actinomycetes with no further differentiation into species.

The community of fungal species on or in barley seeds and malt grain is subject to change through the production process, starting from the developing ear and grain, through harvested grain and ending with the kilned malt. As depicted in Fig. 8.1, the mycobiota is subject to dynamic change in biomass and species spectrum over time. Typical field fungi such as Alternaria spp., Cladosporium spp., Curvularia spp., Drechslera spp., Epicoccum nigrum, Fusarium spp., Microdochium spp., Nigrospora spp., Septoria spp., Trichoderma spp., dominate the spectrum of fungal species because they are able to use the developing grain as substrate without necessarily damaging or killing the embryo. However, many other fungal species representing all major taxonomic groups can be found upon plating of whole barley grains or dilutions of barley meal. The spectrum of fungal species identified from a barley sample varies greatly with time, especially during storage since many species in the genera Aspergillus and Penicillium but also typical xerophiles such as Eurotium spp. or Wallemia sebi only start to develop and multiply after harvest when the water activity of grains decreases to low values during drying and storage. During malt production considerable changes in the prevailing growth conditions mark another fundamental change in the fungal community of the barley grain. High water activities during steeping and high carbon dioxide concentrations during subsequent germination are ideally suited for growth of quite selective species such as Alter- naria spp., Epicoccum nigrum, Fusarium avenaceum, F. graminearum, F. culmorum and F. tricinctum from inside the grain or Geotrichum candidum, Mucor mucedo, Rhizopus oryzae and Rhizopus stolonifer as well as various white and red yeast species on the surface of the grain. Kilning of malt results in heat denaturation of most of the microbiota present on germinated barley. Accordingly, the fungal community undergoes another change in which fast-growing and strongly sporulating Zygomycetes such as Rhizopus ssp. and Mucor spp. as well as yeasts and yeast-like fungi such as Geotrichum candidum and Ramichloridium schulzeri strongly develop under the conditions whereas other members of the typical malting flora are inactivated and eventually killed.

Table 8.1 shows an overview of the spectrum of species frequently encountered on raw barley seeds from the author's own research using culture-based microbiological analysis (myco logical status). For the analysis, grains are surface disinfected by immersing them in a sodium hypochlorite solution (1% active chlorine) for 10 minutes before washing them twice with sterile tap water. One hundred seeds (five per plate) are plated to SNA medium (Nirenberg, 1976; Nirenberg, 1981) containing streptomycin and aureomycin for inhibition of bacterial growth and 2,4-dichlorophenoxyacetic acid (2,4-D) for repression of seed germination. Fungal identification is performed after 14 days of incubation at 17°C in a 12 hours dark/light rhythm with a mixture of white light and UV360 nm light. Light microscopy of the mycelia growing from the seeds is performed at 100-fold magnification directly into the open agar plates. Results show that only a small number of species occur regularly and with relatively high numbers of contaminated seeds per sample. Examples for this group of fungi

Dynamics of the fungal community on barely grain from the field to kilned malt. Redrawn from MQller (1995), with kind permission of Fachverlag Hans Carl GmbH, NQrnberg, Germany

Figure 8.1 Dynamics of the fungal community on barely grain from the field to kilned malt. Redrawn from MQller (1995), with kind permission of Fachverlag Hans Carl GmbH, NQrnberg, Germany.

are Alternaria spp., Epicoccum nigrum, and Fusarium tricinctum which can be found in nearly all samples with relatively low variation in their individual contamination rates. Other species such as Botrytis cinerea, Cladosporium herbarum, Bipolaris sorokini- ana, Fusarium avenaceum, F. equiseti, F. culmorum, F. graminearum, F. poae, Microdochium majus and M. nivale and red yeasts do regularly occur but their numbers show a much higher variation between samples and between years analysed. Others are found unregularly in some years on a varying percentage of samples but fail to be found in others. The spectrum of species detected with the method described above is much more restricted as compared to the spectrum published by Noots et al. (1999). However, it has to be realized that the list of species given in that publication was compiled from the literature and has to be interpreted as the maximum of contamination potentially occurring on a sample of barley. The list of species provided by Flannigan (1969) as a compilation of species occurring on barley seeds with and without surface disinfection and after incubation under various conditions of media and temperature shows much more similarity with the spectrum of species given in Table 8.1. The routine use of surface disinfection and the incubation under fairly low temperatures may provide an explanation for the low numbers of Aspergillus and Penicillium species as well as the low counts and incidence of other typical surface contaminants. In fact, the method used to set up Table 8.1 favours the detection of typical species of field fungi such as Alternaria, Bipolaris, Cladosporium, Epicoccum, Fusarium or Microdochium, which can invade deeper layers of the cereal grain and can thus escape being killed during surface disinfection. The reason for applying a method that strongly selects for field fungi rather than a broader species spectrum by this author is the connection between Fusarium contamination of barley and wheat and the occurrence of gushing in beers produced from such contaminated malt. Niessen et al. (1992) demonstrated a correlation between the percentage of grains in a sample that are contaminated with either Fusarium graminearum, F. cerealis or F. culmorum and the potential of a sample of barley or wheat to cause gushing. Authors set up a maximum level at 3% of grains contaminated by the sum of any of the three species above. It appeared that samples in which this contamination level was exceeded almost always caused gushing in the gushing test described by Donhauser et al. (1990).

Table 8.1 Spectrum of fungal species occurring on selected brewing barley samples from Germany between 2009 and 2014 (n = ± 30 per year) and PCR-based assays for detection and identification

Fungal genus/species

2009

2010

2011

2012

2013

2014

Average (%)

STD (%)

PCR-based detection assays

Acremonium strictum

0.08

0.2

0.14

0.4

0.36

0.20

± 35.30

Doss and Welty (1995), Haugland and Vesper (2000), Meklin et al. (2004)

Alternaria spp.

57.80

73.3

46.6

52.71

59.1

69.1

59.77

± 8.35

Zur et al. (1999, Haugland and Vesper (2000), Johnson et al. (2000)

Aspergillus spp.

0.23

0.04

± 122.47

Mukherjee et al. (2006), Suanthie et al. (2009)

Aureobasidium pullulans

0.17

0.03

± 122.47

Schena et al. (2000a), Schena et al. (2000b), Martini et al. (2009)

Botrytis cinerea

1.17

0.33

0.23

0.86

0.05

0.07

0.45

± 50.82

Rigotti et al. (2002), Brouwer et al. (2003), Gachon and Saindreman (2004)

Chaetomium globosum

0.07

0.6

0.11

± 107.85

Haugland and Vesper (2000)

Cladosporium cladosporioides

1.17

0.21

0.43

0.30

± 76.02

Haugland and Vesper (2000), Zeng et al. (2006)

Cladosporium herbarum

0.92

0.73

1.3

0.79

5.25

4.14

2.19

± 45.31

Haugland and Vesper (2000), Zeng et al. (2006)

Bipolaris sorokiniana

3.30

7

10.1

6.14

5.45

1

5.50

± 28.44

Matusinsky et al. (2010), Aggarwal et al. (2011)

Epicoccum nigrum

16.10

9.38

20.9

23.9

8.85

15.8

15.82

± 122.47

Haugland and Vesper (2000), Martini et al. (2009)

Fusarium acuminatum

0.40

0.14

0.28

0.14

± 19.026

Williams et al. (2002)

Fusarium anthophilum

0.07

0.01

± 62.50

-

Fusarium avenaceum

2.20

1.1

1.4

1.36

0.3

0.86

1.20

± 134.16

Schilling et al. (1996), Turner et al. (1998), Waalwijk et al. (2004)

Fusarium camptoceras

0.07

0.01

± 37.78

-

Fusarium crookwellense

0.07

0.01

± 40.62

Yoder and Christianson (1998)

Fusarium culmorum

0.07

0.38

0.57

0.2

0.5

0.29

± 122.47

Schilling et al. (1996), Nicholson et al. (1998), Mishra et al. (2003), Leisova et al. (2005)

Fusarium equiseti

0.33

0.33

0.31

0.43

0.05

0.64

0.35

± 122.47

Mishra et al. (2003), Nicholson et al. (2004), Jurado et al. (2005), Nicolaisen et al. (2009)

Fusarium graminearum

3.60

2.1

0.77

2.64

0.85

4

2.33

± 27.45

Niessen and Vogel (1997), Nicholson et al. (1998), Doohan et al. (1998), Yang et al. (2008), Yin et al. (2009)

Fusarium langsethiae

0.9

0.07

0.43

1.3

0.64

0.56

± 29.11

Niessen et al. (2004), Wilson et al. (2004), Riazantsev et al. (2008), Nicolaisen et al. (2009)

Fusarium poae

0.30

0.6

2.1

0.93

1.95

2.36

1.37

±31.66

Parry and Nicholson (1996), Yli-Mattila et al. (2004), Niessen et al. (2004), Kulik et al. (2008), Stakheev et al. (2011)

Fusarium sacchari

1.15

0.14

0.22

± 107.32

-

Table 8.1 Continued

Fungal genus/species

2009

2010

2011

2012

2013

2014

Average (%)

STD (%)

PCR-based detection assays

Fusarium sporotrichioides

0.07

0.21

0.05

± 90.83

Kulik et al. (2004), Niessen et al. (2004), Yli-Mattila et al. (2004), Konstantinova and Yli-Mattila (2004), Demeke et al. (2005)

Fusarium solani

0.07

0.07

0.02

± 77.46

Alexandrakis et al. (1998), Jaeger et al. (2000), Lievens et al. (2006)

Fusarium subglutinans

0.08

0.13

0.04

± 80.69

Moller et al. (1999), Mule et al. (2004), Nicolaisen et al. (2009)

Fusarium tricinctum

6.20

2.4

7.2

6.93

4.35

6.36

5.57

± 16.57

Kulik (2008), Nicolaisen et al. (2009), Riazantsev et al. (2008)

Fusarium verticillioides

0.07

0.07

0.02

± 77.46

Beck and Barnett (2003), Mule et al. (2004), Patino et al. (2004), Sanchez-Rangel et al. (2005), Nicolaisen et al. (2009)

Geotrichum candidum

0.2

0.77

0.29

0.75

0.34

±51.97

Nakamura et al. (2007)

Gonatobotrys simplex

0.73

0.07

0.93

0.05

0.14

0.32

± 62.90

-

Harzia acremonioides

0.14

0.02

± 122.47

-

Microdochium majus

3.00

0.33

1.4

1.29

0.6

0.14

1.13

± 46.53

Nicholson et al. (1996), Nicholson and Parry (1996), Glynn et al. (2005)

Microdochium nivale

4.30

0.2

0.23

2.5

0.55

0.21

1.33

± 64.027

Nicholson et al. (1996), Glynn et al. (2005)

Mucor spp.

0.07

0.62

0.1

0.07

0.14

± 82.69

Voigt et al. (1999)

Nigrospora sphaerica

0.27

0.38

0.07

0.05

0.14

0.15

± 48.06

-

Penicillium spp.

0.07

0.62

0.1

0.07

0.14

± 82.69

Pedersen et al. (1997), Mukherjee et al. (2006), Suanthie et al. (2009)

Phoma spp.

0.07

0.01

± 122.47

Keinath et al. (2001)

Ramichloridium schulzeri

1.7

0.29

0.33

± 102.56

-

Rhizopus stolonifer

0.23

0.05

0.05

± 98.59

Nagao et al. (2005)

Red yeast

0.25

0.07

1.3

4.25

0.86

1.12

± 75.59

Garcia et al. (2004), Hierro et al. (2006)

Trichoderma spp.

0.07

0.05

0.02

± 79.06

Hagn et al. (2007)

Ulocladium atrum

0.70

0.14

0.15

0.57

0.26

± 57.78

Haugland and Vesper (2000), Meklin et al. (2004)

White yeast

0.07

0.01

± 122.47

Garcia et al. (2004), Hierro et al. (2006)

No fungal contamination

1.00

3.7

4

1.5

4.2

1.5

2.65

± 27.59

 
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