Investigations on whole cell biocatalysts with application in beverage production and aroma composition

Abstract

In the presented work, investigations of enzyme activities related to the processing of fruits, especially grapes, and the release of aroma-relevant molecules from musts during fermentation are described. For this purpose, among other investigations, a library of more than 2000 yeasts isolated at Hochschule Geisenheim University was screened with respect to their β-glycosidase, pectinase, peptidase and β-lyase activities. Co-fermentation of yeasts with high activities of the mentioned enzyme classes is an attractive alternative to the application of enzyme preparations in winemaking. While no relevant pectinase activities were detected in the isolates studied, high peptidase activities were identified for some Metschnikowia pulcherrima isolates, resulting in greater decreases in the protein signal of musts compared to a selected commercial preparation of the same species. To test organisms for the presence of β-glycosidase activities and to be able to characterize this enzyme activity, four different surrogate substrates are currently mainly used. In another part of the present work, the properties and applicability of these compounds (arbutin, esculin, pNPG and 4-MUG) are compared and the advantages and disadvantages of their use are discussed. All four substrates were found to be suitable for photometric assays. 4-MUG was found to be the most suitable for the intended work due to its high sensitivity, high robustness and ease of use. A direct comparison of these compounds has not been described to date and will facilitate selection for screening approaches and other applications. In addition, the study of the accumulation of the product of hydrolysis of 4-MUG is described, which may provide indications to the localization of β-glycosidases. Following the identification of 4-MUG as the most suitable surrogate substrate, the aforementioned strain library was assayed for β-glycosidase activity. The activity in the supernatant and after cell disruption, as well as the activity of whole cultures at pH 7 and pH 3 were evaluated. Several cell lysates showed high activity, which strongly decreased under wine-typical conditions. Similar results were obtained for the activity in supernatants, with only one strain of the species Rhodotorula mucolaginosa, already known for β-glycosidase activity, showing high activity at low pH values. Currently, most of the known β-glycosidases identified with the above mentioned surrogate substrates are secreted enzymes. These enzymes are easy to find and characterize, but intracellular enzymes with interesting properties may have been previously overlooked. By studying yeasts with cell-associated β-glycosidase activity, the use of whole cells as flavor-enhancing catalysts in winemaking or the processing of other fruit juices should be further established as an alternative to the addition of enzyme preparations, which is currently the predominant practice. Such an approach using whole cells could not only have the advantage of protecting the enzymes against low pH values, but could also lead to different selectivity profiles, as substrate and product transport through the cell membrane is required and whole cells may additionally influence the final flavor composition. The screening identified four yeast strains whose whole cells showed high β-glycosidase activity even at low pH values. Subsequent comparative analyses of the aroma composition in the headspace of assays with whole cells and cell lysates after combination with glycoside extracts indicated a specific influence of intact cells on the released aroma compounds. The highest β-glycosidase activity in must was measured in the experiments with a strain of the species Nakazawaea ishiwadae. Another part of the work presented deals with volatile thiols, which are potent flavor and fragrance compounds found in many fruits and can impart tropical aroma sensations such as passion fruit, guava or blackcurrant. Since these thiols are also characteristic for some wine varieties such as Sauvignon Blanc, their biosynthetic pathways were intensively researched and strategies have been developed to increase their concentrations in winemaking. These include the use of yeast strains with a pronounced ability to release thiols from precursors present in the must. In the work described, a thiol synthesis pathway comprising the corresponding plant and the yeast enzymes was introduced into a laboratory yeast strain, optimized for higher productivity, and characterized in terms of the biosynthetic diversity. In addition to genome integration of the Escherichia coli gene tnaA, which encodes an enzyme with high β-lyase activity, a glutathione synthetase and glutathione-S-transferases were overexpressed. Up to 8.9 μg L-1 3-mercaptohexan-1-ol could be formed with the strain from externally added (E)-2-hexen-1-ol. Well-characterized thiols such as 2-methyl-2-butanthiol, 3-mercapto-3-methylbutan-1-ol, and 8-mercapto-p-menthan-3-one could be formed by feeding the yeast cells with the corresponding alcohol, alkenol, aldehyde or ketone precursors. In addition, several previously undescribed thiol compounds could be synthesized. This concept allows the discovery of thiols with interesting odor properties and could be optimized for a biotechnological production process in the future. Furthermore, the selectivity of yeast- and wine-based glutathione-S-transferases for specific substrates can be analyzed using the described system.