Recent developments in microbial production of high-purity galacto-oligosaccharides.

Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical formulations. Currently, production of GOS involves the enzymatic conversion of lactose by transgalactosylation using ß-galactosidase. The purity of the resulting product is low, typically limited to up to 55% GOS on total carbohydrate basis due to the presence of non-reacted lactose, and the formation of by-products glucose and galactose. In industrial practice high-purity GOS is manufactured by removing the unwanted mono- and disaccharides from raw GOS with simulated moving bed (SMB) chromatography. This purification step is associated with high processing cost that increases the price of pure GOS and limits its marketability. The last decades have witnessed a growing interest in developing competitive biotechnological processes that could replace chromatography. This paper presents a comprehensive review on the recent advancements of microbial GOS purification, a process commonly referred to as selective fermentation or selective metabolism. Purification strategies include: (i) removal of glucose alone or together with galactose by lactose negative yeast species, that typically results in purity values below 60% due to remaining lactose; (ii) removal of both mono- and disaccharides by combining the fast monosaccharide metabolizing capacity of some yeast species with efficient lactose consumption by certain lactose positive microbes, reaching GOS purity in the range of 60-95%; and (iii) the application of selected strains of Kluyveromyces species with high lactose metabolizing activity to achieve high-purity GOS that is practically free from lactose and monosaccharides.

Production of high-purity galacto-oligosaccharides by depleting glucose and lactose from galacto-oligosaccharide syrup with yeasts.

Galacto-oligosaccharides (GOS) are prebiotic compounds, widely used as ingredients in various food, nutraceutical and pharmaceutical products. Enzymatic synthesis of GOS results in low-purity products that contain high amounts of glucose and lactose beside the valuable GOS. In this study, a systematic approach was used to develop yeast-based fermentation strategies to purify crude GOS. Potentially applicable yeast strains were identified based on an extensive search in literature databases followed by a series of laboratory-scale fermentation tests. Single- and two-step fermentation processes were designed for the removal of glucose alone or together with lactose from crude GOS syrup. Single-step fermentation trials with two strains of previously unreported species, Cyberlindnera jadinii NCAIM Y.00499 and Kluyveromyces nonfermentans NCAIM Y.01443, resulted in purified products free of both glucose and ethanol from a crude GOS syrup diluted to 15 and 10 w/v%, respectively. Simultaneous removal of glucose and lactose was achieved by Kluyveromyces marxianus DMB Km-RK in a single-step fermentation process with a yield of 97.5% and final purity of 100%. A two-step fermentation approach was designed to allow conversion of a glucose-free product into a high-purity GOS by removing glucose with C. jadinii Y.00499 in the first step, and lactose by Kluyveromyces lactis DMB Kl-RK in the second step, resulting in a final product with a yield of 100% and a final purity of 92.1%. These results indicate that the selected nonconventional yeasts are promising candidates for the removal of non-GOS components from commercial crude GOS products by selective fermentation.

Antagonistic Effect of Pseudomonas sp. CMI-1 onFoodborne Pathogenic Listeria monocytogenes.

Bacterial isolates derived from food or raw food materials of animal origin were screened for potential antagonistic activity against foodborne pathogenic Listeria monocytogenes. Using the agar spot method, ten out of the 94 tested bacteria showed antilisterial activity. All of the antagonistic isolates identified by sequence analysis as strains of the genus Pseudomonas were able to inhibit the growth of all the examined Listeria species including the ruminal pathogenic L. ivanovii and the opportunistic human pathogenic L. innocua. Pseudomonas sp. CMI-1 had the highest inhibitory effect on the growth of different Listeria strains. Co-culturing studies revealed that the inhibition of L. monocytogenes could not be achieved efficiently. Although the population of the Pseudomonas sp. CMI-1 strain increased by up to 10 orders of magnitude during 2 days of culturing period at 20 °C in the presence of L. monocytogenes, the cell count of the pathogen also increased by approx. 6 orders of magnitude. At the same time, appropriate inhibition of cell-free supernatants generated from 6-day-old cultures of Pseudomonas sp. CMI-1 was observed. The inhibitory compound of this antagonistic strain is presumably a chromopeptide siderophore, whose activity and production can be affected by iron supplementation, and which had an absorption maximum typical of siderophores of fluorescent Pseudomonas species. Production of the antilisterial substance was influenced by the oxygen concentration, as in static cultures the concentration of the siderophore was higher than in shake flask cultures.

[Development of molecular detection of food-borne pathogenic bacteria using miniaturized microfluidic devices]. / Élelmiszer-biztonsági jelentoségu baktériumok molekuláris detektálásának fejlesztése miniatürizált mikrofluidikai eszközök segítségével.

Detection and identification of food-borne pathogenic bacteria are key points for the assurance of microbiological food safety. Traditional culture-based methods are more and more replaced by or supplemented with nucleic acid based molecular techniques, targeting specific (preferably virulence) genes in the genomes. Internationally validated DNA amplification - most frequently real-time polymerase chain reaction - methods are applied by the food microbiological testing laboratories for routine analysis, which will result not only in shortening the time for results but they also improve the performance characteristics (e.g. sensitivity, specificity) of the methods. Beside numerous advantages of the polymerase chain reaction based techniques for routine microbiological analysis certain drawbacks have to be mentioned, such as the high cost of the equipment and reagents, as well as the risk of contamination of the laboratory environment by the polymerase chain reaction amplicons, which require construction of an isolated laboratory system. Lab-on-a-chip systems can integrate most of these laboratory processes within a miniaturized device that delivers the same specificity and reliability as the standard protocols. The benefits of miniaturized devices are: simple - often automated - use, small overall size, portability, sterility due to single use possibility. These miniaturized rapid diagnostic tests are being researched and developed at the best research centers around the globe implementing various sample preparation and molecular DNA amplification methods on-chip. In parallel, the aim of the authors' research is to develop microfluidic Lab-on-a-chip devices for the detection and identification of food-borne pathogenic bacteria.

[Phytochemical and antimicrobial investigation of Epilobium angustifolium L]. / Az Epilobium angustifolium L. (erdei deréce) fitokémiai és antimikrobás hatásnák vizsgálata.

Phytochemical studies of Epilobium angustifolii herba aimed the determination of the active ingredients; antimicrobial activity of the aqueous and ethanolic extracts against human pathogenic bacteria and fungi was also examined. Six flavonoid and 4 phytosterol fractions were identified by thin layer chromatography, while tannins were present in low concentration. It has been shown that not only the ethanolic but also the aqueous extracts had inhibitory effect on certain pathogenic microorganisms, therefore E. angustifolium could be used for the development of external phytotherapic or disinfectant preparations.

Purification and Characterization of a Novel Cold-Active Lipase from the Yeast Candida zeylanoides.

Cold-active lipases have attracted attention in recent years due to their potential applications in reactions requiring lower temperatures. Both bacterial and fungal lipases have been investigated, each having distinct advantages for particular applications. Among yeasts, cold-active lipases from the genera Candida, Yarrowia, Rhodotorula, and Pichia have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from Candida zeylanoides isolated from refrigerated poultry meat are described. Heat-sterilized olive oil was found to be the best lipase biosynthesis inducer, while nonionic detergents were not effective. The enzyme was purified to homogeneity using hydrophobic chromatography and its enzymatic properties were tested. Pure enzyme activity at 7 °C was about 60% of the maximal activity at 27 °C. The enzyme had rather good activity at higher temperatures, as well. Optimal pH of pure lipase was between 7.3 and 8.2, while the enzyme from the crude extract had an optimum pH of about 9.0. The enzyme was sensitive to high ionic strength and lost most of its activity at high salt concentrations. Due to the described properties, cold-active C. zeylanoides lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.

Sulphate metabolism of selenate-resistant Schizosaccharomyces pombe mutants.

Selenate-resistant mutants were obtained from several strains of Schizosaccharomyces pombe. The obtained mutants all belonged to the same genetic complementation group. They were low in sulphate uptake activity and in ATP sulphurylase activity. They grew on medium containing sulphite, thiosulphate, cysteine or glutathione but not methionine as the sole source of sulphur. From these results, the mutants were concluded to carry mutations in the ATP sulphurylase gene. Inability of the mutants to utilize methionine as a sulphur source is rationalized by the absence of the reverse transsulphurylation pathway in this organism; wild type strains must utilize methionine as a sulphur source after it is degraded to give rise to sulphate.

Cloning of the ATP sulphurylase gene of Schizosaccharomyces pombe by functional complementation.

The ATP sulphurylase gene of Schizosaccharomyces pombe has been cloned by complementation of cysteine auxotrophy of a selenate-resistant mutant, which supposedly had a defect in ATP sulphurylase. A sulphate nonutilizing (cysteine auxotrophic) and selenate-resistant mutant of S. pombe was transformed with a wild-type S. pombe genomic library and sulphate-utilizing clones were isolated. The open reading frame encoding the ATP sulphurylase enzyme was found to be responsible for the restoration of sulphate assimilation. Transformants became as sensitive for selenate as the wild-type strain and produced a comparable amount of ATP sulphurylase as the prototrophic strains. The cloned ATP sulphurylase gene (sua1) proved to be an efficient selection marker in an ARS vector, when different isogenic or nonisogenic S. pombe selenate-resistant mutants were used as cloning hosts. Complementation of sua1- mutations by sua1-bearing multicopy vectors functions as a useful dual positive and negative selection marker. The cloned sua1 gene also complemented the met3 (ATP sulphurylase deficient) mutation in Saccharomyces cerevisiae.

Characterization of Ccw7p cell wall proteins and the encoding genes of Saccharomyces cerevisiae wine yeast strains: relevance for flor formation.

The specific flavour of Sherry-type wines requires aromatic compounds produced as by-products of the oxidative metabolism of yeasts that are able to form a biofilm (flor) at the wine surface. A similar yeast pellicle develops on the surface of 'Tokaji Szamorodni', one of the traditional Hungarian botrytized wines, during maturation. In this work, patterns of biotinylated cell wall proteins extracted from film-forming and nonfilm-forming Saccharomyces cerevisiae strains were compared. It was found that all the tested 23 film-forming 'Szamorodni' yeast strains had a decreased size of the Ccw7/Hsp150 protein, one of the members of the Pir-protein family. Sequencing of the encoding genes revealed that the strains were lacking three out of the 11 repeating sequences characteristic to this protein family. One of the film-forming strains contained CCW7 alleles of different length, which was generated by intragenic tandem duplication of a sequence containing two repetitive domains. Unlike the film-forming strains, 16 nonfilm-forming wine yeasts isolated from a different botrytized wine, 'Tokaji Aszu', showed pronounced polymorphism of the CCW7 locus. It is highly probable that the modified Ccw7 protein does not contribute to the increased hydrophobicity of film-forming strains but it may influence molecular reorganization of the cell wall during stress adaptation.

Cell wall ultrastructure of flocculent and non-flocculent Schizosaccharomyces pombe strains. Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure.

Scanning and transmission electron microscopic studies revealed the presence of slime-like, amorphous material on the surface of Schizosaccahromyces pombe RIVE 4-2-1 cells, independently, whether they were in flocculated or in non-flocculated state. Close contact of the adjacent cells via the merging outermost cell wall layers was found, however, only in the case of floc formation, which was induced by cultivating the cells in the presence of 6% (v/v) ethanol. Irreversible loss of the flocculation ability of the cells by treatment with proteinases suggests that proteinaceous cell surface molecules as lectins contribute to the cell-to-cell interaction during flocculation. Both proteinase K and pronase treatments removed a distinct outer layer of the cell wall, which indicated that the protein moieties of the phosphogalactomannan outer surface layer has a crucial role in the maintenance of cell wall integrity. In the case of lysing enzyme treatment the removal of the outermost layer was also observed as the first step of the cell wall digestion, while driselase treatment resulted in almost complete digestion of the cell wall.

From yeast genetics to biotechnology.

Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S. cerevisiae. Consequent mutation and hybridization of heterothallic S. cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map. Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains. Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains [1, 2]. In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g. sugar utilization, flocculation and flavor production in brewing yeast strains [3] or in the improvement of ethanol producing S. cerevisiae strains [4]. Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement [5]. Importance of yeasts in biotechnology is enormous. This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S. cerevisiae is undoubtedly the most important species among them. New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas. Without decreasing the importance of S. cerevisiae, numerous other yeast species, e.g. Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern fermentation biotechnology [6]. Developments in yeast genetics, biochemistry, physiology and process engineering provided bases of rapid development in modern biotechnology, but elaboration of the recombinant DNA technique is far the most important milestone in this field. Other molecular genetic techniques, as molecular genotyping of yeast strains proved also very beneficial in yeast fermentation technologies, because dynamics of both the natural and inoculated yeast biota could be followed by these versatile DNA-based techniques.

Genetics of sulphate assimilation in Schizosaccharomyces pombe (a short review).

Sulphur plays an important role in yeasts, especially in the biosynthesis of methionine and cysteine. The inorganic sulphur source, sulphate, is taken up by the cells via the sulphate-permease(s). After its transport, it is activated and subsequently reduced to sulphide or serves as a donor for sulphurylation reactions. Selenate anion (SeO4(2-)), which has the same metabolic pathway as sulphate, is toxic for the cells of Schizosaccharomyces pombe. We isolated selenate resistant mutants which cannot utilize sulphate, therefore they need organic sulphur source for growth. One of the selenate resistant mutants was successively transformed with S. pombe genomic libraries and the gene complementing the selenate resistance was identified as that of coding for the ATP-sulphurylase enzyme.

Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events.

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.