Diversity of yeasts and yeast-like fungi

Four industrial malting runs were thoroughly investigated (Paper III). Figure 13 shows the yeast growth in industrial maltings. We also studied the effect of growth temperature on the yeast counts. As shown in Figure 13, yeasts in the malting ecosystem were capable of growing at 15 °C as well as at 25 °C. Our results were in agreement with those of Petters et al. (1988), who also found that the yeasts in the malting ecosystem were favoured by their ability to grow at low temperatures prevailing in steeping and germination. The malting ecosystem also harboured yeasts capable of growing at 37 °C (Figure 13). However, greater variation in the number of thermotolerant yeasts was observed within batches compared to the populations at 15 or 25 °C. These yeasts probably originated from the malting equipment, and batch to batch variation in the process environment and in malting procedures could explain the observed fluctuation. It has been shown that a specific microbial community develops in each malting plant and it also has significant effects on the properties the final product (O’Sullivan et al. 1999, Petters et al. 1988).

As seen from Figure 13, kilning appeared to have little effect on the viable yeast counts. Only tenfold reduction in yeast counts was observed during kilning. In fact the first hours of kilning before the temperature breakthrough, especially in the top layers of the grain bed, appeared to be rather favourable for yeast growth.

Under normal environmental conditions, the vegetative yeast cells are rapidly inactivated by temperatures of 60–65 °C. (Fleet 1992.) This study revealed that a large proportion of the yeast community was composed of encapsulating yeasts, which could explain the high number of survivors in the kilned malt. In the malting ecosystem the microbial cells embedded in thick biofilms were well protected. Schwarz et al. (1995) also reported a large increase in the ergosterol content during the early hours of kilning, indicating that fungal growth (both yeasts and filamentous fungi) was accelerated. It is clear that a significant amount of fungal metabolites such as enzymes is formed during this stage, which may later have an impact during the mashing stage. In addition, synthesis of harmful fungal metabolites such as mycotoxins has been reported during kilning (Schwarz et al. 1995). Therefore, kilning can also be regarded as an important step with respect to microbiological safety.

Figure 13. Growth of yeasts during the industrial scale malting process.

•) Yeasts cultivated at 15 °C, ■) yeasts cultivated at 25 °C and ▲) yeasts cultivated at 37 °C. Open small symbols are the minimum values and closed small symbols are the maximum values detected in determinations.

The main goal of this study (Paper III) was to obtain an overall picture of the yeasts in the industrial malting ecosystem. A total of 136 malting samples were collected from four industrial processes. More than 700 yeast isolates were first discriminated with PCR-fingerprinting using an oligonucleotide primer (M13) targeting simple repetitive DNA sequences (microsatellites). This protocol has been widely applied in yeast typing and allows the discrimination of yeast species even at the subspecies level (Loureiro 2000). Yeasts representing different fingerprint types were then identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene (Fell et al. 2000, Kurtzman & Robnett 1998).

We detected 25 species of ascomycetous yeasts belonging to eight genera and 18 species of basidiomycetous yeasts belonging to six genera from the malting trials with Saana barley from the 2001 crop (Table 10). Previously only 10 ascomycetous and 6 basidiomycetous yeast species have been reported from

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0

0 20 40 60 80 100 120 140 160 180 200

malting time, h

Steep Germination Kilning

detection limit log CFU/g

barley and malting samples (Douglas & Flannigan 1988, Flannigan 1969, Flannigan et al. 1982, Flannigan & Dickie 1972, Kottheimer & Christensen 1961, Noots et al. 1999, Petters et al. 1988, Tuomi et al. 1995, Tuomi &

Rosenqvist 1995).

Table 10. Yeast species detected in the industrial malting ecosystem. Identified strains were deposited in the VTT Culture Collection and their 26S rRNA gene sequences were deposited in GenBank under the numbers shown in Tables 2 and 3 in Paper III.

Genera Species Ascomycetous

Candida anglica, cylindracea, fermentati, intermedia, natalensis, pararugosa, picinguabensis, saitoana, sake, silvae, solani, Candida sp. I and II ¹⁾

Clavispora lusitaniae Galactomyces geotrichum Geotrichum silvicola

Hanseniaspora clermontiae/meyri²⁾, uvarum Issatchenkia orientalis

Pichia anomala, fabianii, fermentans, guilliermondii Saccharomyces exiguus

Williopsis californica Yeast-like fungi

Aureobasidium pullulans Exophiala dermatidis Basidiomycetous

Bulleromyces albus

Cryptococcus albidosimilis, curvatus, hungaricus, macerans, magnus, victoriae, wieringae, Cryptococcus sp. I, II, III and IV ³⁾ Filobasidium globisporum

Rhodotorula glutinis, pinicola Sporobolomyces roseus, ruberrimus Trichosporon brassicae

1) Two sets of Candida isolates did not match closely enough to any sequences present at the time in the database.

2) Species cannot be separated by D1/D2 sequencing.

3) Four groups of undescribed Cryptococcus species, indicated as Cryptococcus sp. I–IV, were found on the basis of D1/D2 sequences (Paper III, Table 3).

All the identified yeast species were detected at least at a level of 10⁴–10⁵ cfu/g.

Some minor species may have been overlooked in the present study and thus the yeast diversity in the malting ecosystem could be even greater. Basidiomycetous yeasts dominated the yeast community of barley (Paper III, Table 4).

Furthermore, they were frequently detected during the first days of malting. The growth of basidiomycota was favoured by the low temperatures during steeping.

Many basidiomycetous species have temperature optima below 20 °C (Deak 1991).

In contrast to basidiomycetous species, ascomycetous yeasts dominated at the end of germination and during the first hours of kilning. We found 20 different ascomycetous yeasts in the samples taken after 5 h of kilning, whereas only five basidiomycetous yeasts were detected in the same samples (Paper III, Table 4).

The occurrence of ascomycetous yeasts was obviously due to their ability to grow better at the higher temperatures than basidiomycetous yeasts.

This study provided a clear indication of the vast yeast diversity in the malting ecosystem. It is obvious that even more yeast heterogeneity could be expected due to the differences between barley crops as well as between industrial practices in different locations. Even some potentially novel species were found in the malting ecosystem. The unidentified isolates have been subjected to further characterization. To confirm that the strains represent different species, multigene sequence analysis is required (Kurtzman & Robnett 2003). Analysis of combined gene sequences such as internal transcribed spacer regions of the rRNA genes (ITS), the actin gene and mitochondrially encoded genes will provide more information of the genetic relationships than partial analysis of the 26S rRNA gene (Daniel & Meyer 2003, Fell et al. 2000, Kurtzman and Robnett 2003).