Ústav chemie potravin a biotechnologií
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- ItemAbundance of G-Quadruplex Forming Sequences in the Hepatitis Delta Virus Genomes(American Chemical Society, 2024-01-09) Brázda, Václav; Válková, Natálie; Dobrovolná, Michaela; Mergny, Jean-LouisHepatitis delta virus (HDV) is a highly unusual RNA satellite virus that depends on the presence of hepatitis B virus (HBV) to be infectious. Its compact and variable single-stranded RNA genome consists of eight major genotypes distributed unevenly across different continents. The significance of noncanonical secondary structures such as G-quadruplexes (G4s) is increasingly recognized at the DNA and RNA levels, particularly for transcription, replication, and translation. G4s are formed from guanine-rich sequences and have been identified in the vast majority of viral, eukaryotic, and prokaryotic genomes. In this study, we analyzed the G4 propensity of HDV genomes by using G4Hunter. Unlike HBV, which has a G4 density similar to that of the human genome, HDV displays a significantly higher number of potential quadruplex-forming sequences (PQS), with a density more than four times greater than that of the human genome. This finding suggests a critical role for G4s in HDV, especially given that the PQS regions are conserved across HDV genotypes. Furthermore, the prevalence of G4-forming sequences may represent a promising target for therapeutic interventions to control HDV replication.
- ItemRaman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms(BMC, 2023-12-18) Dzurendová, Simona; Olsen, Pernille Margrethe; Byrtusová, Dana; Tafintseva, Valeria; Shapaval, Volha; Horn, Svein Jarle; Kohler, Achim; Szotkowski, Martin; Márová, Ivana; Zimmermann, BorisBackground: Monitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups. Results: The system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R-2) in the 0.94-0.99 and 0.89-0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles. Conclusions: The FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.
- ItemLiposomes Affect Protein Release and Stability of ITA-Modified PLGA-PEG-PLGA Hydrogel Carriers for Controlled Drug Delivery(AMER CHEMICAL SOC, 2023-12-22) Kadlecová, Zuzana; Sevriugina, Veronika; Lysáková, Klára; Rychetský, Matěj; Chamradová, Ivana; Vojtová, LucyFat grafting, a key regenerative medicine technique, often requires repeat procedures due to high-fat reabsorption and volume loss. Addressing this, a novel drug delivery system uniquely combines a thermosensitive, FDA-approved hydrogel (itaconic acid-modified PLGA-PEG-PLGA copolymer) with FGF2-STAB, a stable fibroblast growth factor 2 with a 21-day stability, far exceeding a few hours of wild-type FGF2's stability. Additionally, the growth factor was encapsulated in "green" liposomes prepared via the Mozafari method, ensuring pH protection. The system, characterized by first-order FGF2-STAB release, employs green chemistry for biocompatibility, bioactivity, and eco-friendliness. The liposomes, with diameters of 85.73 +/- 3.85 nm and 68.6 +/- 2.2% encapsulation efficiency, allowed controlled FGF2-STAB release from the hydrogel compared to the unencapsulated FGF2-STAB. Yet, the protein compromised the carrier's hydrolytic stability. Prior tests were conducted on model proteins human albumin (efficiency 80.8 +/- 3.2%) and lysozyme (efficiency 81.0 +/- 2.7%). This injectable thermosensitive system could advance reconstructive medicine and cosmetic procedures.
- ItemThe Effect of Oil-Rich Food Waste Substrates, Used as an Alternative Carbon Source, on the Cultivation of Microalgae-A Pilot Study(MDPI, 2023-06-21) Sniegoňová, Pavlína; Szotkowski, Martin; Holub, Jiří; Sikorová, Pavlína; Márová, IvanaMicroalgae are mostly phototrophic microorganisms present worldwide, showcasing great adaptability to their environment. They are known for producing essential metabolites such as carotenoids, chlorophylls, sterols, lipids, and many more. This study discusses the possibility of the mixotrophic abilities of microalgae in the presence of food waste oils. The utilization of food waste materials is becoming more popular as a research subject as its production grows every year, increasing the environmental burden. In this work, waste frying oil and coffee oil were tested for the first time as a nutrition source for microalgae cultivation. Waste frying oil is produced in large amounts all over the world and its simple purification is one of its greatest advantages as it only needs to be filtered from leftover food pieces. Coffee oil is extracted from waste spent coffee grounds as a by-product. The waste frying oil and coffee oil were added to the basic algal media as an alternative source of carbon. As a pilot study for further experimentation, the effect of oil in the medium, algal adaptability, and capability to survive were tested within these experiments. The growth and production characteristics of four algae and cyanobacteria strains were tested, of which the strain Desmodesmus armatus achieved exceptional results of chlorophyll (8.171 & PLUSMN; 0.475 mg/g) and ubiquinone (5.708 & PLUSMN; 0.138 mg/g) production. The strain Chlamydomonas reindhartii showed exceptional lipid accumulation in the range of 30-46% in most of the samples.
- ItemConversion of Mixed Waste Food Substrates by Carotenogenic Yeasts of Rhodotorula sp. Genus(MDPI, 2023-04-13) Szotkowski, Martin; Plhalová, Žaneta; Sniegoňová, Pavlína; Holub, Jiří; Chujanov, Oleg; Špačková, Dominika; Blažková, Jana; Márová, IvanaThe consequence of the massive increase in population in recent years is the enormous production of mainly industrial waste. The effort to minimize these waste products is, therefore, no longer sufficient. Biotechnologists, therefore, started looking for ways to not only reuse these waste products, but also to valorise them. This work focuses on the biotechnological use and processing of waste oils/fats and waste glycerol by carotenogenic yeasts of the genus Rhodotorula and Sporidiobolus. The results of this work show that the selected yeast strains are able to process waste glycerol as well as some oils and fats in a circular economy model and, moreover, are resistant to potential antimicrobial compounds present in the medium. The best-growing strains, Rhodotorula toruloides CCY 062-002-004 and Rhodotorula kratochvilovae CCY 020-002-026, were selected for fed-batch cultivation in a laboratory bioreactor in a medium containing a mixture of coffee oil and waste glycerol. The results show that both strains were able to produce more than 18 g of biomass per litre of media with a high content of carotenoids (10.757 +/- 1.007 mg/g of CDW in R. kratochvilovae and 10.514 +/- 1.520 mg/g of CDW in R. toruloides, respectively). The overall results prove that combining different waste substrates is a promising option for producing yeast biomass enriched with carotenoids, lipids, and beta-glucans.