Antifungal potential of yeasts derived from the

Our study also revealed that yeasts derived from the malting ecosystem had a antifungal potential (Paper IV). In vitro screening with the plate assay indicated that several ascomycetous strains belonging to the species A. pullulans, C. sake, C. saitoana, G. geotrichum, P. anomala and P. guilliermondii showed antifungal activity against field and storage fungi (Paper IV, Table 1).

Table 12. Antifungal potential of selected yeasts and yeast-like fungi against Fusarium-fungi in a plate-screening assay. - No inhibition, + suppression of mould growth.

VTT-D Aureobasidium pullulans D-041014 Candida sake C-95520 C. saitoana C-04524 G. silvicola D-04559 Pichia anomala C-04564 P. anomala C-04565 P.guilliermondii C-04568 Cryptococcus albidus C-92012 Cr. albidosimilis C-04508 Cr. curvatus C-04536 Cr. magnus C-04540 Rhodotorula pinicola C-04571

F. avenaceum 80141 + + + + + + + + + + + +

F. cerealis 96601 + + + + + + + - + + - - F. culmorum 80148 - - + + + + + - - - - - F. equiseti 82087 - + + + - + - - - - - - F. graminearum 82169 - - + + + + + - - - - - F. graminearum 95470 + - + + + + + - + - - - F. langsethiae 03931 - - + + + + - - - -

F. oxysporum 80134 - - - + + + - - -

F. poae 76038 - - + - + + + - - - - -

F. sambucinum 77056 - - + + + + - - - - F. sporotrichioides 82175 - - - + + + + - - - F. sporotrichioides 72014 + - + + + + - + - - - -

F. tricinctum 96607 - - - + + + - - -

The main emphasis was on the suppression of Fusarium growth. All the yeast strains tested could prevent the overgrowth of F. avenaceum D141 in the plate-screening assay, whereas F. oxysporum D134 and F. tricinctum D607 strains were restricted only by G. silvicola D559 and P. anomala strains C564 and C565 (Table 12). P. anomala C565 strain was selected for malting experiments in order to verify the antifungal potential of malt-derived yeast in malting with naturally infested barley.

Our results are supported by previous investigations which also reported the antifungal activity of these yeasts in other applications (Fredlund et al. 2004, Passoth et al. 2005, Saligkarias et al. 2002, Schena et al. 2003, Wisniewski et al.

1991). Several biocontrol yeasts are nowadays commercially available. For example yeast strains belonging to Candida oleophila, Cryptococcus albidus, and Metschnikowia fructicola are commercialized and have been successfully applied to prevent pre- and post-harvest fungal diseases of fruits and vegetables (Boekhout & Robert 2003, Janisiewicz & Korsten 2002). P. anomala J121 strain has been applied to control the spoilage moulds during storage of high moisture feed grains (Druvefors et al. 2002, Passoth et al. 2005). Geotrichum candidum, also known as an IFBM malting yeast, has been developed for inhibiting fungal growth and mycotoxin production in malting (Boivin & Malanda 1997, Boivin 2002).

The effects of one potential biocontrol agent, P. anomala C565, were also examined in malting with naturally contaminated barley exhibiting gushing potential. To our knowledge this is the first report that shows the effects of P.

anomala against fusaria in malting and the consequent effect on the overall malt quality. P. anomala occurs naturally in cereals and is classified as safe (biosafety level 1 microorganisms) (Druvefors 2004). This study revealed that the addition of P. anomala C565 (isolated from an industrial malting process) into the steeping was highly suppressive to Fusarium and Mucor-fungi (Figure 14). We showed that P. anomala inhibited the production of fungal hydrophobic proteins during malting and prevented gushing (Paper IV, Figure 3, Table 2). Hydrophobins are among the most important structural proteins found in filamentous fungi (Ebbole 1997). Hydrophobins are produced in response to changes in the environment and are linked to the attachment of fungi to plant surfaces (Wessels 1997). Fungal hydrophobins also act as gushing inducers of beer, although the production of gushing factors in malting is still largely an unknown phenomenon

(Haikara et al. 2000, Sarlin et al. 2005). It is well known that intensive Fusarium growth is a part of the normal malting process. However, to our knowledge the production of gushing factors occurs only rarely in industrial malting processes.

The results of the present study indicate that some suppression of Fusarium growth and hydrophobin production probably occurs in normal industrial practice with the aid of the indigenous yeasts community.

Although the mode of action of the antifungal activity remains to be revealed, the results indicated that P. anomala C565 competed for space with fusaria. As a fast growing organism, P. anomala colonized the outer layers of barley and suppressed the adherence of fungal contaminants to the barley surface during steeping. Antifungal action of antagonistic yeasts is often due to several mechanisms and hitherto no single mechanism has been shown to be responsible for the complete antimicrobial action. The mechanisms are poorly understood, especially in such complex ecosystems. Competition for nutrients and space has often been suggested as the main mode of action. In addition, the antifungal action of antagonistic yeasts includes induction of the plant defence system, production of lytic enzymes such as β-1-3 glucanase and chitinase, which degrade the fungal cell wall, or secretion of antimicrobial compounds such as killer proteins (Janisiewicz & Korsten 2002, Masih & Paul 2002, Passoth &

Schnürer 2003). Druvefors et al. (2002) suggested that the antifungal effect of P. anomala was due to the synergistic action of ethyl acetate and ethanol produced by Pichia in oxygen-limited environment. This study revealed that ethyl acetate was indeed detected in the gaseous atmosphere of the malting drum in P. anomala-treated samples, which might partly explain the antifungal action against fusaria.

A) B)

Control (A), P. anomala C565 (B) (IV).

P. anomala C565 had no significant effect on the bacterial communities or on grain germination. We showed that P. anomala utilized the grain volatile metabolites as a substrate for growth, without disturbing the grain’s normal germination process (Paper IV, Table 3, Figure 5). Furthermore, the final malts were well modified. However, P. anomala C565 treatment tended to retard mash filterability when added to the steeping water. As a strongly antagonistic organism P. anomala suppressed the growth of other yeasts and filamentous fungi and led to a decreased production of microbial β-glucanase and xylanase, which could partly explain the reduced filtration rate (Paper IV, Table 4).

Furthermore, the slight increase in wort viscosity in Pichia-containing samples indicated the presence of high-molecular weight polysaccharides. Kreisz et al.

(2001) reported that malt-derived yeast polysaccharides such as mannan and glycogen may have a significant impact on the haze levels of filtered beer.

Therefore, precautions must be taken when selecting biocontrol agents to malting applications. The negative impact of P. anomala on filtration performance may limit its use in malting applications alone. In the present study a rather high inoculum level was used (10⁶ yeast cells / gram of barley) in steeping. The inoculum level and stage in the malting process need further investigations. However, this study clearly confirmed the previous findings (Druvefors et al. 2002, Fredlund et al. 2004, Passot & Schnürer 2003, Petersson

& Schnürer 1998, Petersson et al. 1999) that P. anomala strains have great antifungal potential and can be used in cereal-based processes to inhibit the growth of spoilage fungi.

4.7 Tailoring malt properties with combined