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Structure optimisation of biopigment prodigiosin from Serratia marcescens ATCC 27117 and antimicrobial and anticancer properties of novel halogenated derivatives
Jelena Lazić, 2022, doctoral dissertation

Abstract: Prodigiosins (PGs) are a class of bacterial secondary metabolites with remarkable biological activities and colour. In this study, optimised fermentative production of prodigiosin (PG) using waste processed meat as a substrate has been achieved to levels of 83.1 ± 3.0 mg/L from a commercially available Serratia marcescens ATCC 27117 strain within 12 h. Methods were established for the reliable PG extraction from both the bacterial cell pellet and the culture supernatant, while gravitation column chromatography was used to obtain pure bacterial PG. The structure of the isolated PG was optimised by environmentally acceptable oxidative bromination reactions, obtaining mono- and dibrominated derivatives (PG-Br and PG-Br2). Chemical structures were confirmed by structural characterisation using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), showing that PG-Br is a mixture of two monobrominated isomers in approximately equal ratios, while PG-Br2 was afforded as a pure derivative. PG and its brominated derivatives (Br-derivatives) showed anticancer potential with half-maximal inhibitory concentration (IC50) values ranging from 0.62 to 17.00 μg/mL on four tested cancer cell lines (A549 lung, A375 skin, MDA-MB-231 breast, HCT116 colon) and an induction of early apoptosis, but low selectivity against healthy cell lines (MRC-5 lung fibroblasts and HaCaT skin keratinocytes). All three PG, PG-Br and PG-Br2 compounds did not affect roundworms (Caenorhabditis elegans) at concentrations up to 50 μg/mL. However, an improved toxicity profile of Br-derivatives in comparison to the parent PG was observed in vivo using zebrafish (Danio rerio) model system, when 10 μg/mL applied at 6 h post fertilisation caused death rate of 100, 30 and 0% by PG, PG-Br and PG-Br2, respectively, which is a significant finding for further structural optimisations of bacterial PGs.
Keywords: prodigiosin, Serratia marcescens, meat waste, halogenation, novel derivatives, anticancer activity, apoptosis, zebrafish embryotoxicity
Published in RUNG: 21.10.2022; Views: 1546; Downloads: 31
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Novel analytical approaches in quality and safety control in production of fermented beverages : dissertation
Jelena Topić, 2022, doctoral dissertation

Abstract: The exploitation of microorganisms for fermentation goes back centuries. Two types of fermentation are usually used in the winemaking process – alcoholic fermentation and malolactic fermentation. Nowadays, inoculated fermentations with the use of starter cultures are commonly used in order to produce wine with more consistent quality. However, wines can lack in flavour complexity, so scientists and the industry are constantly looking for new and improved starters that can be adapted to different types of wine. In this work we focused on the development and implementation of novel analytical methods for wine quality control. In the course of method development native yeasts and lactic acid bacteria isolates were characterized for wine starter properties. We focused on the determination of biologically active compounds that determine wine quality and safety. Yeasts can influence wine colour through their adsorption capacity and synthesis of stable colour pigments pyranoanthocyanins and lactic acid bacteria can produce biogenic amines which can have adverse detrimental health effects on sensitive consumers when they are present in wines.
Keywords: Saccharomyces yeasts, non-Saccharomyces yeasts, pyranoanthocyanins, thermal-lens spectrometry, lactic acid bacteria, biogenic amines, thin layer chromatography, dissertations
Published in RUNG: 18.02.2022; Views: 2682; Downloads: 120
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Determination of biogenic amines formation by autochthonous lactic acid bacteria from ‘Refošk’ grapes using different analytical methods
Jelena Topić, Lorena Butinar, Martina Bergant Marušič, Dorota Korte, Branka Mozetič Vodopivec, 2022, original scientific article

Abstract: Different analytical methods were tested and optimized for the determination of four biogenic amines (BA) histamine, putrescine, cadaverine and tyramine produced by grape-associated lactic acid bacteria (LAB). Autochthonous LAB were isolated from ‘Refošk’ grapes belonging to Slovenian-Italian Karst region as they represent a potential pool of beneficial LAB starter bacteria for improving the typicality and quality of wine. Six isolated strains were screened by multiplex PCR, of which four strains were positive for BA-forming genes (MKBT-49, MKBT-282, MKBT-568, MKBT-570). The production of BA was evaluated by thin-layer chromatography (TLC), high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and enzymatic method. The HPLC-DAD results showed that strain MKBT-49 (tyrdc+) had high tyramine production (386.6 ± 0.14 mg/L), which was confirmed by TLC. The ability to produce putrescine was confirmed in strain MKBT-282 by PCR, HPLC-DAD (16.4 ± 1.72 mg/L), and TLC. Histamine-producing ability was detected in strain MKBT-570, with a concentration below the limit of detection of the HPLC-DAD (<0.2 mg/L), while the other two methods were not sensitive enough for confirmation. This study shows that the production of BA can detected in native LAB and that relatively simple method such as TLC can be used effectively for the initial screening.
Keywords: grapes, wine-like matrices, biogenic amines, autochthonous lactic acid bacteria, analytical methods
Published in RUNG: 03.01.2022; Views: 1730; Downloads: 7
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Determination of biogenic amine-forming potential of naturally occuring lactic acid bacteria (LAB) strains isolated from the grapes of Vitis vinifera cv. Refošk grape variety
Jelena Topić, Lorena Butinar, Martina Bergant Marušič, Dorota Korte, Branka Mozetič Vodopivec, 2020, published scientific conference contribution abstract

Abstract: Biogenic amines are naturally occurring compounds that have been reported in variety of food, such as fish, meat, cheese, vegetables, and wines. When biogenic amines are formed by microorganisms such as lactic acid bacteria (LAB) they may have adverse effect on human health. The most common biogenic amines found in foodstuff are histamine, tyramine, putrescine and cadaverine. LAB are used as starters in the production of several foods and beverages. In wines they are used in malolactic fermentation, where the conversion of malic acid to lactic acid occurs. The aim of this work was to determine biogenic amine-forming capacity of indigenous LAB strains isolated from grapes which were PCR positive and screening medium positive for biogenic amines. Three methods for the confirmation of LAB biogenic amine-forming potential were developed and compared: HPLC-DAD and TLC methods both using derivatization agent dansyl chloride and enzymatic method using horseradish peroxidase (HRP) and diamine oxidase (DAO) enzymes. The applicability of methods was tested on the three known biogenic amine-producing LAB strains: 0006, 9809, 9906 (IOEB, France). The limit of detection (LOD) of the methods was <1 mg/L for HPLC-DAD, 5 mg/L for TLC and 25 mg/L for enzymatic method. Although the LODs between the proposed methods differed, all of the three methods confirmed BA-forming potential of the three strains. Furthermore, the proposed methods were applied for the analysis of three indigenous strains: MKBT-282 (BA+), MKBT-307 (BA-) and MKBT-325 (BA+). The positive or negative BA-forming potential was confirmed with all of the three proposed methods.
Keywords: lactic acid bacteria, biogenic amines, histamine, grape
Published in RUNG: 10.07.2020; Views: 3021; Downloads: 0
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