Modulation of fungal biofilm physiology and secondary product formation based on physico-chemical surface properties.

Relative to the amount of knowledge concerning bacterial biofilms, little is known about the impact of physico-chemical properties of support material on fungal biofilm adhesion and physiology. In the field of industrial fermentation, large-scale production of low-cost fungal secondary product is a challenging area of research. In the present work, the effect of physico-chemical surface properties of five different materials (Teflon, glass, Viton™ rubber, silicon rubber, and stainless steel) on the production of class II hydrophobins (HFBI and HFBII) from Trichoderma reesei (HFB2a-2) and Trichoderma harzianum) was evaluated. Two culture systems (shake flask and drip flow reactor (DFR)) were used in this study to promote biomass growth and the production of hydrophobins. Furthermore, the effect of physico-chemical surface properties (hydrophobicity, surface energy) and surface texture (roughness) of support material on the initial colonization and attachment of the fungal biofilm was evaluated. Maximum biofilm productivity was obtained using Viton™ rubber for T. reesei and Viton™ rubber and stainless steel as support materials for T. harzianum. Scanning electron microscope (SEM) revealed that fungal biofilm adhesion was higher on the rough hydrophobic Viton rubber surface as compared to the smooth hydrophobic Teflon surface. Initial colonization initiated because of surface irregularities and holes in the material as hyphal filaments. Moreover, compared to traditional submerged fermentation, a significant increase in biofilm productivity for both strains (T. reesei, T. harzianum) in all five materials was obtained.

Polydopamine imprinted magnetic nanoparticles as a method to purify and detect class II hydrophobins from heterogeneous mixtures.

Hydrophobins are one of the most active surface active proteins in nature, with an amphiphilic nature and the ability to self-assembly in elastic monolayers, the possible applications in industry are continuously increasing. However, production and purification of these proteins still remains a tedious process. We introduce here the use of polydopamine as imprinter polymer to create specific magnetic nanoparticles for the recognition of Hydrophobin HFBII from Trichoderma reesei. The protein was molecularly imprinted to magnetic nanoparticles to facilitate its specific detection and purification from liquids or carbonated beverages in the presence of other proteins. The resulting magnetic nanoparticles were successfully imprinted adsorbing till 77,4µg of HFBII hydrophobin per miligram of nanoparticles. The adsorption capacity of the imprinted nanoparticles was also tested for specificity using a mixture of five different proteins and peptides. A slight cross interaction was observed when proteins of similar molecular weight to HFBII were used. With larger proteins and peptides the interaction was very low. with other class II Hydrophobins the interaction was very similar as to HFBII.

Characterization of the recombinant Brettanomyces anomalus ß-glucosidase and its potential for bioflavouring.

AIM: Plant materials used in the food industry contain up to five times more aromas bound to glucose (glucosides) than free, unbound aromas, making these bound aromas an unused flavouring potential. The aim of this study was to identify and purify a novel ß-glucosidase from Brettanomyces yeasts that are capable of releasing bound aromas present in various food products. METHODS AND RESULTS: We screened 428 different yeast strains for ß-glucosidase activity and are the first to sequence the whole genome of two Brettanomyces yeasts (Brettanomyces anomalus and Brettanomyces bruxellensis) with exceptionally high ß-glucosidase activity. Heterologous expression and purification of the identified B. anomalus ß-glucosidase showed that it has an optimal activity at a higher pH (5·75) and lower temperature (37°C) than commercial ß-glucosidases. Adding this B. anomalus ß-glucosidase to cherry beers and forest fruit milks resulted in increased amounts of benzyl alcohol, eugenol, linalool and methyl salicylate compared to Aspergillus niger and Almond glucosidase. CONCLUSIONS: The newly identified B. anomalus ß-glucosidase offers new possibilities for food bioflavouring. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to sequence the B. anomalus genome and to identify the ß-glucosidase-encoding genes of two Brettanomyces species, and reports a new bioflavouring enzyme.

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

AIMS: The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. METHODS AND RESULTS: A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. CONCLUSIONS: Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. SIGNIFICANCE AND IMPACT OF THE STUDY: The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

Bioflavoring and beer refermentation.

Various techniques are used to adjust the flavors of foods and beverages to new market demands. Although synthetic flavoring chemicals are still widely used, flavors produced by biological methods (bioflavors) are now more and more requested by consumers, increasingly concerned with health and environmental problems caused by synthetic chemicals. Bioflavors can be extracted from plants or produced with plant cell cultures, microorganisms or isolated enzymes. This Mini-Review paper gives an overview of different systems for the microbial production of natural flavors, either de novo, or starting with selected flavor precursor molecules. Emphasis is put on the bioflavoring of beer and the possibilities offered by beer refermentation processes. The use of flavor precursors in combination with non-conventional or genetically modified yeasts for the production of new products is discussed.

Yeast flocculation: what brewers should know.

For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.