Jiřina Omelková

Monitoring of yeast population isolated during spontaneous fermentation of Moravian wine

In enology, yeasts play an important role in the characteristics of the final product. They are predominant in the biochemical interaction with components of must. Rapid identification of the yeast population is necessary for fermentation process monitoring and for obtaining a good quality wine. The main goal of this study was the isolation and characterisation of the yeast microbial community naturally present on grape berries, leaves and occurring during the spontaneous fermentation process of the white wine Veltlin green from the South Moravian region, Czech Republic. The results, based on PCR-RFLP of the 5.8S-ITS region of rDNA, PCR-fingerprinting using microsatellite oligonucleotide primers (GAG)5, (GTG)5, (GAC)5, and M13 primer, showed great diversity of the yeast population. Including grape berries and fermented must, the following yeast species were identified: Hanseniaspora uvarum, Aureobasidium pullulans, Metschnikowia pulcherrima, Torulaspora delbrueckii, a number of Pichia species such as P. fermentans, P. membranifaciens, P. kluyveri, also Sporidiobolus salmonicolor, Rhodosporidium toruloides, Rhodotorula mucilaginosa, Rhodotorula glutinis as well as Saccharomyces cerevisiae and Saccharomyces bayanus. Monitoring of the yeast strains during the wine fermentation process of traditional Moravian wine can contribute to the improvement of wine quality.

Oligogalacturonate hydrolase from carrot roots.

The presence of multiple forms of enzyme with terminal action pattern on pectate was evaluated in the protein mixture obtained from carrot roots. The form with pH optimum 3.8 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates). Its molecular mass, isoelectric point, glycosylation as well as cleavage of pectate from nonreducing end corresponded to an exopolygalacturonase [EC 3.2.2.67]. The affinity of this enzyme to the substrates increased with the increasing degree of polymerization, and the difference was observed only in the maximal ratio of catalysis of oligomeric and polymeric substrates. Sterical hindrance for substrates with more than six ᴅ-galactopyranuronic acid units is supposed and an oligogalacturonate hydrolase rather than exopolygalacturonase is considered.

Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains.

In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

The Effect of Processing of Polycaprolactone Films on Degradation Process Initiated by Aspergillus Oryzae Lipase

Poly(ϵ-caprolactone) (PCL) films with Mn = 18 kDa obtained by compression molding (CM) or solution casting (SC) were subjected to Aspergillus oryzae (AO) lipase action in a phosphate buffer at pH 7 at 37°C. The appearance of randomly oriented cracks on the surface of incubated PCL films accompanied by a decrease of the weight-average molecular weight (Mw) by 10% was observed after 42 days. The increase of crystallinity and surface morphology pattern of PCL samples exposed to AO lipase action supported the fact that the degradation proceeded in the amorphous phase of the aged films. SC films were degraded faster, probably due to better accessibility of ester bonds in the amorphous phase of spherulites.

Purification and biochemical characterization of polygalacturonases produced by Aureobasidium pullulans.

The extracellular polygalacturonases produced by Aureobasidium pullulans isolated from waters of the Danube river were partially purified and characterized. The pH optima of polygalacturonases produced in the first phases of cultivation (48 h) and after 10 d as well as their optima of temperature, thermal stabilities, molecular masses, isoelectric points, action pattern and ability to cleave polymeric and oligomeric substrates were compared. Polygalacturonases with a random action pattern (random cleavage of pectate forming a mixture of galactosiduronides with a lower degree of polymerization) [EC 3.2.1.15] were produced only in the first phases of growth, while exopolygalacturonases [EC 3.2.1.67] with a terminal action pattern (cleavage of pectate from the nonreducing end forming d-galactopyranuronic acid as a product) were found during the whole growth. The main enzyme form with a random action pattern was glycosylated and its active site had the arrangement described previously for the active site of polygalacturonase of phytopathogenic fungi.

Production of Geotrichum candidum polygalacturonases via solid state fermentation on grape pomace

Geotrichum candidum CCY 16-1-29 (teleomorph Galactomyces geotrichum) is able to grow and produce polygalacturonase of remarkable activities on pectin or grape pomace as a sole carbon source. The highest activities of extracellular enzymes were found on the third and the seventh day of cultivation. After extraction and precipitation, polygalacturonases produced in these cultivation periods were characterized. Production of multiple forms of polygalacturonase was observed in both cultivation periods. Two major forms, polygalacturonase with random action pattern (endo-PGase, EC 3.2.1.15) and oligogalacturonate hydrolase (exoPGase, exopolygalacturonase preferring oligogalacturonides as substrates), as well as numerous minor forms were detected by IEF-PAGE using the print technique detection. EndoPGase was identified by mass spectrometry. The major forms have similar isoelectric points (below pH 6.0) and pH optima (4.6 and 4.8, respectively). pH optimum of 4.6 was associated with exoPGase and that of 4.8 with endoPGase. Both enzymes were stable after freeze-drying and storage at 4°C. EndoPGase had molecular mass of about 29 kDa (36 kDa by SDS-PAGE) as determined by gel filtration, temperature optimum of about 45°C and it was stable only below 35°C. Molecular mass of exoPGase was about 50 kDa, its temperature optimum was about 60°C, and it was stable to 60°C. Optimal substrate for exoPGase was a pentamer, for endoPGase it was a pectate. Values of Km for optimal substrate reached the values of 11.4 × 10−5 M for for exoPGase and 6.6 × 10−5 M for endoPGase. Pectin methylesterase as another pectolytic enzyme was also identified by mass spectrometry.

Pectate hydrolases of parsley (Petroselinum crispum) roots.

The presence of various enzyme forms with terminal action pattern on pectate was evaluated in a protein mixture obtained from parsley roots. Enzymes found in the soluble fraction of roots (juice) were purified to homogeneity according to SDS-PAGE, partially separated by preparative isoelectric focusing and characterized. Three forms with pH optima 3.6, 4.2 and 4.6 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates) while the form with pH optimum 5.2 was a typical exopolygalacturonase [EC 3. 2.1.67] with relatively fast cleavage of polymeric substrate. The forms with pH optima 3.6, 4.2 and 5.2 were released from the pulp, too. The form from the pulp with pH optimum 4.6 preferred higher oligogalacturonates and was not described in plants previously. The production of individual forms in roots was compared with that produced by root cells cultivated on solid medium and in liquid one.

The Impact of Graphite Source and the Synthesis Method on the Properties of Graphene Oxide

The purpose of this study is to insight more thoroughly into the structural properties of graphene oxide (GO) in particular of the type generated by Hummers method in relation with graphite precursor originating from different sources. The systematic study using elemental analysis (EA), thermogravimetric analysis (TGA), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) was performed to reveal the changes of GO microstructure. The results revealed considerable influence of both the original graphite source and isolation procedure of GO by lyophilisation. Further, maintaining of GO in a form of aqueous colloidal dispersion is crucial for preservation of its unique properties. SEM results revealed the occurrence of associated GO blocks formed form identically oriented planes.

Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate preference of a plant exopolygalacturonase because it clearly preferred the substrates with degree of polymerization about 10. This form was separated from the others, purified and characterized. Enzyme exhibited sharp pH optimum corresponding to pH 4.7, molecular mass 53.5 kDa, and isoelectric point 5.3. It was stable at 50°C in 2-h assay and had optimum of temperature at 60°C (activation energy being 37.0 kJ/mol). The interaction with concanavalin A indicated the glycosylation of enzyme. Substrates were cleaved from the non-reducing end.

The life style of Aureobasidium pullulans and the multiple forms of its polygalacturonase

The life style of Aureobasidium pullulans on pectin medium and its production of extracellular polygalacturonases are closely related. Polygalacturonases with random action pattern (EC 3.2.1.15) were formed in the first phases of cultivation, whereas exopolygalacturonases (EC 3.2.1.67) with terminal action pattern on pectin were produced during the whole growth of this yeast-like fungus. The production and inactivation of individual enzyme forms during cultivation were strongly dependent on the pH value of the pectin medium. Various kinds of stress can support the prolongation of the phase of endo-acting enzyme production, as well as the increase of their activity.