Ivan Milovanović

Development of a rapid resolution HPLC method for the separation and determination of 17 phenolic compounds in crude plant extracts

AbstractRapid Resolution HPLC/DAD method, on a 1.8 µm, 4.6×50 mm column, was developed to enable a rapid separation of a mixture of 17 compounds, which consisted of hydroxybenzoic acids, hydroxycinnamic acids, flavones, flavonols, flavanone, flavonol-glycoside and antraquinone, in a single run, within 22 minutes. The developed method is precise, accurate and sensitive enough for simultaneous quantitative evaluation of major compounds in crude and hydrolyzed extracts of parsley, buckthorn, mint, caraway and birch. In order to overcome the inability to quantify all the phenolic compounds present in the samples caused by lack of external standards, HPLC approaches for the total phenolic content estimation based on sum of all integrated peak areas were made. These results were compared with the total phenolic content determined by Folin-Cioacalteu method. Although the correlation between the series of data was not significant (p<0.05), the difference between the results of total phenolic content obtained spectrophotometrically and by HPLC was not high in the case of parsley, buckthorn and mint extract. Regarding the obtained results, the HPLC approach could serve as an excellent tool for total phenolic content estimation, without the need for complete identification of the individual compounds.

Characterization of Byproducts Originating from Hemp Oil Processing

Valorization of hemp seed meal, a byproduct of hemp oil processing, was performed by measuring the distribution of nutritional and antinutritional compounds in different hemp seed meal fractions. According to chemical composition, two cotyledon-containing fractions (>180 and <180 μm) were significantly richer in protein (p < 0.05) (41.2% ± 0.04% and 44.4% ± 0.02%, respectively), lipid (15.1% ± 0.02% and 18.6% ± 0.04%, respectively), and sugar content (4.96% ± 0.11% and 3.46% ± 0.08%, respectively) in comparison to the hull-containing fractions (>350 and >250 μm), which were significantly richer in crude fiber content (29.5% ± 0.04% and 21.3% ± 0.03%, respectively). The free radical scavenging capacity (IC50) of fraction extracts increased (p < 0.05) with increasing mean particle size (from 17.18 ± 0.59 to 5.29 ± 0.30 mg/mL). Cannabisin B and N-trans-caffeoyltyramine were the most abundant phenolic compounds in the hull fractions (from 267 ± 15.9 to 287 ± 23.1 mg/kg), while cotyledon fractions had higher content of catechin (from 313 ± 12.4 to 744 ± 22.2 mg/kg) and p-hydroxybenzoic acid (from 124 ± 6.47 to 129 ± 8.56 mg/kg (P < 0.05). Well-balanced ω-6 to ω-3 fatty acid ratio (3:1) was determined in all fractions. Antinutrients (trypsin inhibitors, phytic acid, glucosinolates, and condensed tannins) were mostly located in the cotyledon fractions. These findings indicate that the separation of hemp seed meal into different fractions could be used to concentrate valuable target compounds and consequently facilitate their recovery.

Determination of Volatile Organic Compounds in Selected Strains of Cyanobacteria

Microalgal biomass can be used in creating various functional food and feed products, but certain species of microalgae and cyanobacteria are known to produce various compounds causing off-flavour. In this work, we investigated selected cyanobacterial strains of Spirulina, Anabaena, and Nostoc genera originating from Serbia, with the aim of determining the chemical profile of volatile organic compounds produced by these organisms. Additionally, the influence of nitrogen level during growth on the production of volatile compounds was investigated for Nostoc and Anabaena strains. In addition, multivariate techniques, namely, principal component analysis (PCA) and hierarchical cluster analysis (HCA), were used for making distinction among different microalgal strains. The results show that the main volatile compounds in these species are medium chain length alkanes, but other odorous compounds such as 2-methylisoborneol (0.51–4.48%), 2-pentylfuran (0.72–8.98%), β-cyclocitral (0.00–1.17%), and β-ionone (1.15–2.72%) were also detected in the samples. Addition of nitrogen to growth medium was shown to negatively affect the production of 2-methylisoborneol, while geosmin was not detected in any of the analyzed samples, which indicates that the manipulation of growth conditions may be useful in reducing levels of some unwanted odor-causing components.

Potential of Pleurotus ostreatus Mycelium for Selenium Absorption

The aim of this study was to evaluate the effect of high selenium (Se) concentrations on morphophysiological and ultrastructural properties of Pleurotus ostreatus. Mycelium growth was good in media enriched with 5.0, 10.0, and 20.0 mg L−1 of Se, concentration of 500.0 mg L−1 strongly inhibited growth, and 1000.0 mg L−1 was the minimum inhibitory concentration. Contrary to thin-walled, hyaline, branched, and anastomized hyphae with clamp-connections in the control, at Se concentrations of 100.0 and 500.0 mg L−1, they were noticeably short, frequently septed and branched, with a more intensive extracellular matrix, and without clamp-connections. At high Se concentrations, hyphae with intact membrane, without cellular contents, with a high level of vacuolization, and with numerous proteinaceous bodies were observed. Biomass yield ranged between 11.8 g L−1, in the control, and 6.8 g L−1, at an Se concentration of 100.0 mg L−1, while no production was detected at a concentration of 500.0 mg L−1. Se content in the mycelia reached a peak (938.9 μg g−1) after cultivation in the medium enriched with Se at the concentration of 20.0 mg L−1, while the highest absorption level (53.25%) was found in the medium enriched with 5.0 mg L−1 Se.

Induction of wheat straw delignification by Trametes species.

Wheat straw is the major crop residue in European countries which makes it the most promising material for bioconversion into biofuels. However, cellulose and hemicellulose are protected with lignin, so delignification is an inevitable phase in lignocellulose processing. The organisms predominantly responsible for its degradation are white-rot fungi and among them Trametes species represent promising degraders due to a well-developed ligninolytic enzyme system. Although numerous studies have confirmed that low molecular weight compounds can induce the production and activity of ligninolytic enzymes it is not clear how this reflects on the extent of delignification. The aim of the study was to assess the capacity of p-anisidine and veratryl alcohol to induce the production and activity of Mn-oxidizing peroxidases and laccases, and wheat straw delignification by six Trametes species. Significant inter- and intraspecific variations in activity and features of these enzymes were found, as well as differences in the potential of lignocellulose degradation in the presence or absence of inducers. Differences in the catalytic properties of synthesized enzyme isoforms strongly affected lignin degradation. Apart from enhanced lignin degradation, the addition of p-anisidine could significantly improve the selectivity of wheat straw ligninolysis, which was especially evident for T. hirsuta strains.

Antioxidant and Antifungal Potential of Pleurotus ostreatus and Agrocybe cylindracea Basidiocarps and Mycelia

Basidiocarps of Pleurotus ostreatus and Agrocybe cylindracea are characterized by high nutritional value and numerous medicinal activities, though bioactivities of their mycelia have not been sufficiently studied. The aim of the study was to evaluate antioxidant and antifungal potentials of P. ostreatus and A. cylindracea basidiocarp extracts, as well as those obtained from mycelia cultivated in common synthetic medium and media enriched with various agro-industrial residues. The free radical scavenging activity of the extracts was determined spectrophotometrically, based on DPPH• reduction while antifungal potential was studied by a microdilution method. The highest level of DPPH• scavenging ability was obtained by an extract of P. ostreatus mycelium cultivated in wheat bran-enriched medium, while control medium favoured the antioxidant potential of A. cylindracea mycelium. Phenol compounds were the main carriers of antioxidant activity that was demonstrated by high coefficients for correlations between total phenol contents in extracts and level of DPPH• scavenging (0.94 for P. ostreatus extracts and 0.91 for A. cylindracea extracts). Only the extracts of A. cylindracea basidiocarp and control mycelium, at a concentration of 1.33 mg/mL, inhibited the growth of Microsporum gypseum and Aspergillus flavus, while basidiocarp extract at 1.67 mg/mL also had fungicidal effect against M. gypseum. These results revealed a considerable antioxidant potential of submergedly-cultivated mycelium which showed higher antioxidant activity than basidiocarp extracts. This is very important because significant amounts of mycelium biomass could be obtained more easily, cheaper and in a more controllable way than basidiocarps cultivation.

Antigenotoxic Effect of Trametes spp. Extracts against DNA Damage on Human Peripheral White Blood Cells

Trametes species have been used for thousands of years in traditional and conventional medicine for the treatment of various types of diseases. The goal was to evaluate possible antigenotoxic effects of mycelium and basidiocarp extracts of selected Trametes species and to assess dependence on their antioxidant potential. Trametes versicolor, T. hirsuta, and T. gibbosa were the species studied. Antigenotoxic potentials of extracts were assessed on human peripheral white blood cells with basidiocarp and mycelium extracts of the species. The alkaline comet test was used for detection of DNA strand breaks and alkali-labile sites, as well as the extent of DNA migration. DPPH assay was used to estimate antioxidative properties of extracts. Fruiting body extracts of T. versicolor and T. gibbosa as well as T. hirsuta extracts, except that at 20.0 mg/mL, were not genotoxic agents. T. versicolor extract had at 5.0 mg/mL the greatest antigenotoxic effect in both pre- and posttreatment of leukocytes. The mycelium extracts of the three species had no genotoxic activity and significant antigenotoxic effect against H2O2-induced DNA damage, both in pre- and posttreatment. The results suggest that extracts of these three species could be considered as strong antigenotoxic agents able to stimulate genoprotective response of cells.

Antibacterial activity of domestic Balkan donkey milk toward Listeria monocytogenes and Staphylococcus aureus

The aim of this study was to investigate the antibacterial activity of raw milk from Domestic Balkan donkey breed toward Listeria monocytogenes and Staphylococcus aureus. Examination of antibacterial activity was performed in artificially contaminated milk samples by monitoring the changes of count of viable cells of tested bacteria during 8 hours of incubation at 38°C. Lysozyme and fatty acids contents were also determined in donkey milk. The obtained results indicated inhibitory effect of donkey milk toward both tested bacteria. The lysozyme content in the analyzed milk samples was ranged from 0.67 to 3.54 g/L. The most abundant fatty acids with known antibacterial activity toward Gram positive bacteria were linoleic, lauric and oleic acid.

Effect of Origanum heracleoticum L. essential oil on food-borne Penicillium aurantiogriseum and Penicilium chrysogenum isolates

Molds are ubiquitously distributed in nature and their spores can be found in the atmosphere even at high altitudes. The difficulty of controlling these undesirable molds, as well as the growing interest of the consumers in natural products, have been forcing the industry to find new alternatives for food preservation. The modern trends in nutrition suggest the limitation of synthetic food additives or substitution with natural ones. Aromatic herbs are probably the most important source of natural antimicrobial agents. Origanum heracleoticum L. essential oil has been known as an interesting source of antimicrobial compounds to be applied in food preservation. In the this work, we have investigated the effect of essential oil obtained from O. heracleoticum on growth of six isolates of Penicillium aurantiogriseum and four isolates of Penicillium chrysogenum isolated from meat plant for traditional Petrovacka sausage (Petrovska klobasa) production. The findings reveal that the essential oil of O. heracleoticum provides inhibition of all of fungal isolates tested. O. heracleoticum L. essential oil exhibited higher antifungal activity against the isolates of P. chrysogenum than the isolates of P. aurantiogriseum. O. heracleoticum essential oil showed a MIC value ranging from 25 to 100 μL/mL. The fungi cultivated in the medium with higher concentration of essential oil showed certain morphological changes. The alterations included lack of sporulation and loss of pigmentation.

Antioxidant and Physicochemical Properties of Hydrogen Peroxide-Treated Sugar Beet Dietary Fibre.

The aim of the present work was to examine if hydrogen peroxide treatment of sugar beet fibre that aimed at improving its physicochemical properties would impair its antioxidant potential. Three different sugar beet fibres were obtained from sugar beet – non‐treated fibre (NTF) from sugar beet cossettes extracted with sulphurous acid, treated fibre (TF) from NTF treated with hydrogen peroxide in alkaline solution and commercially available Fibrex®. The antioxidant activity of extractable and non‐extractable fibre fractions in ethanol/water mixture (80:20, v/v) of three fibre samples was estimated. Non‐extractable fractions obtained after alkaline treatment of investigated fibres were much higher in phenolic compounds and possessed higher antioxidant potential than extractable fractions. Ferulic acid was proven to be the dominant phenolic acid. Regarding both extractable and non‐extractable fractions, Fibrex® had the highest antioxidant activity in chemical tests, while NTF was superior in comparison with TF. Based on the results of Caco‐2 cells‐based test, all non‐extractable fractions possessed potential for reactive oxygen species inhibition. Regarding the extractable fractions, only the TF manifested this effect.Copyright