Yeasts in malting,with special emphasis on <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> (synonym <Emphasis Type="Italic">Pichia anomala</Emphasis>)

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

Identity, beer spoiling and biofilm forming potential of yeasts from beer bottling plant associated biofilms

Wild yeasts were isolated from process surfaces of two breweries. In total, 41 strains were obtained and differentiated by cultivation on CuSO₄ or crystal violet containing selective media, by fatty acid profiling and by a restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene. The restriction analysis showed the highest differentiating capacity and resulted in eleven groups. These groups were identified by the API ID 32 C kit or by sequencing the D1/D2 region of the 26S rRNA gene. Most of the wild yeasts were identified as Saccharomyces cerevisiae (46% of all isolates) and Candida pelliculosa (anamorph: Pichia anomala) (24%). No obvious differences were detected between the two breweries. While all of the S. cerevisiae isolates were able to grow in beer, only six out of 10 C. pelliculosa strains were able to tolerate this substrate. However, most of the C. pelliculosa strains showed biofilm formation in a microplate assay, but none of the S. cerevisiae isolates. Therefore, it is assumed that the former species is involved in attachment and primary biofilm formation on beer bottling plants, while S. cerevisiae is a late colonizer of a preformed biofilm but increased the beer spoiling potential of the biofilm.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

<Emphasis Type="Italic">Pichia anomala</Emphasis>: cell physiology and biotechnology relative to other yeasts

Pichia anomala is a most interesting yeast species, from a number of environmental, industrial and medical aspects. This yeast has been isolated from very diverse natural habitats (e.g. in foods, insects, wastewaters etc.) and it also exhibits wide metabolic and physiological diversity. Some of the activities of P. anomala, particularly its antimicrobial action, make it a very attractive organism for biological control applications in the agri-food sectors of industry. Being a ‘robust’ organism, it additionally has potential to be exploited in bioremediation of environmental pollutants. This paper provides an overview of cell physiological characteristics (growth, metabolism, stress responses) and biotechnological potential (e.g. as a novel biocontrol agent) of P. anomala and compares such properties with other yeast species, notably Saccharomyces cerevisiae, which remains the most exploited industrial microorganism. We await further basic knowledge of P. anomala cell physiology and genetics prior to its fuller commercial exploitation, but the exciting biotechnological potential of this yeast is highlighted in this paper.     

Nutrient Effects on Biocontrol of Penicillium roqueforti by Pichia anomala J121 during Airtight Storage of Wheat

The biocontrol yeast Pichia anomala inhibits the growth of a variety of mold species. We examined the mechanism underlying the inhibition of the grain spoilage mold Penicillium roqueforti by the biocontrol yeast P. anomala J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of complex medium or glucose did not significantly influence the yeast cell numbers in the silos, whether in the presence or absence of P. roqueforti. Mold growth was not influenced by the addition of nutrients, if cultivated without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose addition to P. anomala-inoculated treatments. Our results suggest that neither competition for nutrients nor production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by P. anomala in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites, most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar amendments enhance the biocontrol effect of P. anomala suggests novel ways of formulating biocontrol yeasts.     

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast <Emphasis Type="Italic">Pichia anomala</Emphasis> J121

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> in grain biopreservation

Cereal grain is a major component of food and feed in large parts of the world. The microbial flora on cereal grains may interfere with hygiene and storage stability, palatability and bioavailability of minerals and proteins may depend on the composition of the microbial population. Therefore, it is of primary interest to control the microbial species present on cereal grain. Inoculation of the biocontrol yeast Pichia anomala to cereal feed grain improved feed hygiene by reduction of moulds and Enterobacteriaceae, and enhanced the nutritional value by increasing the protein content and reducing the concentration of the antinutritional compound phytate. P. anomala strains showed a high phytase activity, for some strains also considerable extracellular phytase activity was observed. A certain maximum in biomass concentration was never exceeded indicating cell density induced growth inhibition of P. anomala.     

The use of yeasts for biological control of the plant pathogen Sclerotinia sclerotiorum

Yeasts are promising biological control agents(BCAs) for a number of plant diseases. Studieswere carried out to evaluate various adjuvantsand nutrients for their ability to supportgrowth of a yeast BCA (Cryptococcusalbidus). Hydroxyethylcellulose (HEC) andinvert emulsions were found to stimulate growthof C. albidus in vitro. Severalcommercial spray adjuvants were compatible withC. albidus although they did not markedlystimulate growth. Other adjuvants were lethalto the yeast. In controlled environmentand field trials, the yeasts C. albidusand Pichia anomala provided low levels ofcontrol of white mould, a disease of bean (Phaseolus vulgaris) caused by the fungus Sclerotinia sclerotiorum. However, they weregenerally inferior in performance when comparedto either the biocontrol fungus Epicoccumnigrum or to the fungicide iprodione.     

Fluorescent Viability Stains to Probe the Metabolic Status of Aflatoxigenic Fungus in Dual Culture of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Pichia anomala</Emphasis>

The metabolic activity of the aflatoxigenic fungus, Aspergillus flavus co-cultured with the biocontrol yeast, Pichia anomala was examined using several viability stains. Both the FUN-1 stain and the combined use of DiBAC₄(5) with CDFA-AM stains were applied in this study. The results suggest that the ATP-generating system in A. flavus was inactivated as the ratio of yeasts to fungi increased in the dual culture. A decrease in hyphal membrane potential and esterase activity was substantiated by the combined stains of DiBAC₄(5) and CDFA-AM. Reduced metabolic function in conjunction with cell wall damage of A. flavus hindered the growth and biomass production of this fungus. Viability stains such as FUN-1 and DiBAC₄(5) with CDFA-AM may assist in elucidating the biocontrol mechanism by allowing for the visualization of the antagonistic effect of yeast species on target fungi in situ, as well as for screening potent biocontrol yeast agents against fungal pathogens.     

Identification and characterization of yeasts in brem, a traditional Balinese rice wine

Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae(35 strains), Candida glabrata(six strains), Pichia anomala(three strains) and Issatchenkia orientalis(seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.     

<Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> in the sourdough microbial ecosystem

We previously found that Wickerhamomyces anomalus (formerly Hansenula anomala, Pichia anomala) was the second most frequently isolated yeast in Belgian artisan bakery sourdoughs and that the yeast dominated laboratory sourdough fermentations. Such findings are of interest in terms of the advantage of W. anomalus over other commonly encountered sourdough yeasts and its potential introduction into the sourdough ecosystem. Here, we provide a brief overview of current knowledge on yeast ecology and diversity in sourdough in the context of the potential natural habitat of W. anomalus. Insight into the population structure of W. anomalus was obtained by comparing internal transcribed spacer rDNA sequences of selected sourdough isolates with publicly available database sequences.     

Improvement in the biocontrol of postharvest diseases of apples with the use of yeast mixtures

Mixtures of yeasts were tested for theirability to control Penicillium expansum andBotrytis cinerea on Red Delicious apple fruits. The occurrence of synergistic or antagonisticinteractions between yeast strains in differentmixtures was also evaluated. Two strains ofRhodotorula (R. glutinis SL 1 and R. glutinisSL 30) and two strains of Cryptococcus (C. albidus SL 43 and C. laurentii SL 62) were selected fordeveloping yeasts mixtures.The R. glutinis SL 1–R. glutinis SL 30 mixtureexhibited a lower effectiveness than eachstrain alone, against both moulds. Othermixtures (R. glutinis SL 1–C. albidus SL 43 and R. glutinis SL 30–C. albidus SL 43) showedsynergism against P. expansum but not against B. cinerea. The R. glutinis SL 1–C. laurentii SL62 mixture was the only mixture which showedsynergism against gray mould. There was not anymixture, which showed high effectivenessagainst both moulds at the same time. Differentresults could be explained by the dynamics ofthe population of the yeasts.By using yeast mixtures, it was possible toimprove biocontrol without increasing theamount of antagonists applied. The synergismobserved could be useful in enhancingbiological control.     

Fungi,Aflatoxins, Fumonisin B<Subscript>l</Subscript> and Zearalenone Contaminating Sorghum-based Traditional Malt,Wort and Beer in Botswana

Brewing and consumption of traditional beer have social–economic significance in most African countries including Botswana. Traditional sorghum malt, wort, and beer samples were collected from three villages around Gaborone, Botswana. Forty-six malt samples were analyzed for fungi on three different media and developing colonies were subcultured for identification. Rhizopus, Fusarium, Mucor, and Aspergillus were the most common genera isolated. Out of the 46 malt samples, 72% contained Rhizopus stolonifer, 63% Fusarium verticillioides (syn. Fusarium moniliforme), and 37% Aspergillus flavus. Although Aspergillus flavus was isolated from malt samples, aflatoxins (B₁, B₂, G₁, and G₂) were not detected in any of the samples analyzed. When the malt, wort, and beer samples were analyzed for fumonisin B_l and zearalenone, fumonisin B₁ was detected in 3 malt samples, with concentrations ranging from 47 to 1316 μg/kg, while zearalenone was detected in 56%, 48% and 48% of the malt, wort and beer samples, respectively. Zearalenone concentration in samples ranged from 102 to 2213 μg/kg in malt, 26 to 285 μg/l in wort and 20 to 201 μg/l, in beer. Zearalenone carry-over from wort to beer ranged from 23 to 403%. Therefore, although aflatoxins and fumonisin B₁ do not appear to be major contaminants, zearalenone is common and could pose a potential problem in traditional beer in Botswana.     

Sexual forms of yeasts in clinical samples

The sexual or teleomorphic state of yeasts has only been described in a few clinically involved species, mainly those of the Saccharomycetaceae family. With the aim of gathering information on their incidence in human pathology, a study has been made of a total of 2,135 strains isolated from clinical samples and cultivated in McClary agar. From these, 8 strains in teleomorphic state were identified: Kluyveromyces marxianus [1], Pichia anomala [2], Pichia farinosa [1], Pichia membranaefaciens [1] and Saccharomyces cerevisiae [3]. The two strains of P. anomala were responsible for fungemia; K. marxianus and the two strains of S. cerevisiae produced vaginitis; the other strains were oral cavity colonizers.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> J121: a 30-year overnight near success biopreservation story

Thirty years ago, the ascomycetous yeast Pichia anomala strain J121 was isolated from moist wheat grain stored under conditions of restricted air access. Early observations indicated that an inverse relationship existed between mould and P. anomala colony forming units in grain. This yeast strain was later found to have strong antifungal properties in laboratory, pilot and farm studies with high-moisture wheat under malfunctioning airtight storage. P. anomala had the highest inhibitory activity of 60 yeast species evaluated against the mould Penicillium roqueforti. It also demonstrated strong inhibitory effects against certain Gram-negative bacteria. P. anomala J121 possesses a number of physiological characteristics, i.e. capacity to grow under low pH, low water activity and low oxygen tension and ability to use a wide range of carbon and nitrogen sources, enabling it to act as an efficient biopreservative agent. The biocontrol effect in grain was enhanced by addition of glucose, mainly through formation of the volatile antimicrobial ethyl acetate. Animal feeding trials with P. anomala J121 inoculated grains, fed to chickens and beef cattle, demonstrated that mould control observed in vitro in small scale laboratory experiments could be extended to large scale farm trials. In addition, no adverse effects on animal weight gain, feed conversion, health or behaviour were observed. We have now studied P. anomala J121 biology, ecology and grain preservation ability for 30 years. Over this period, more than 40 scientific publications and five PhD theses have been written on different aspects of this yeast strain, extending from fundamental research on metabolism, genetics and molecular biology, all the way to practical farm-scale level. In spite of the well documented biopreservative ability of the yeast, it has to date been very difficult to create the right constellation of technical, agricultural and biotechnical industries necessary to reach a commercial launch of a P. anomala J121 based biopreservation system. Additionally, the complications caused by a complex EU regulatory system remain a significant barrier to practical applications.     

The impact of Fusarium culmorum infection on the protein fractions of raw barley and malted grains

Contaminating fungi, such as Fusarium species, produce metabolites that may interfere with normal barley grain proteolysis pattern and consequently, affect malt and beer quality. Protein compositional changes of an initial mixture of 20 % Fusarium culmorum infected and 80 % noninfected mature barley grains and respective malt are reported here. Proteolytic activity of infected barley grains (IBG) and respective malt, with controls (uninfected grains), were characterized using protease inhibitors from each class of this enzyme, including metallo-, cysteine, serine, and aspartic proteases, as well as uninhibited protease fractions. The proteins were extracted according to the Osborne fractionation and separated by size exclusion chromatography. Additionally, two-dimensional (2D) gel electrophoresis (GE) was used to analyze hydrophobic storage proteins isolated from the control and IBG. Analyses revealed that F. culmorum IBG had a twofold increase of proteolytic activity compared to the control sample, which showed an increase in all protease classes with aspartic proteases dominating. Infected and control malt grains were comparable with cysteine proteases representing almost 50 % of all proteolytic enzymes detected. Protein extractability was 31 % higher in IBG compared to the control barley. The albumin fraction showed that several metabolic proteins decreased and increased at different rates during infection and malting, thus showing a complex F. culmorum infection interdependence. Prolamin storage proteins were more hydrophobic during barley fungal infection. F. culmorum interfered with the grain hydrolytic protein profile, thereby altering the grain's protein content and quality.     

Spoilage yeasts from Patagonian cellars: characterization and potential biocontrol based on killer interactions

Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage yeasts.     

Yeasts in an industrial malting ecosystem

The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37°C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of α-amylase, β-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.