Tomaž Polak

Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids.

The compounds 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), 4-vinylsyringol (4-VS) and 4-vinylcatechol (4-VC) were prepared by thermal decarboxylation of the corresponding hydroxycinnamic acids p-coumaric, ferulic, sinapic and caffeic acid, respectively. For confirmation of the synthesised products LC-MS followed by NMR analysis was used. To evaluate their antioxidant potential, their reducing power and efficiency in scavenging the alkylperoxyl radical generated in an emulsion system, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and the superoxide anion radical (O2(-)) were determined. All tested 4-vinyl derivatives revealed weaker antioxidant activity in a homogeneous polar medium than the corresponding phenolic acids. In the emulsion system the activity for 4-vinyl derivatives was higher than was the activity of their corresponding phenolic acids, with 4-VG as the most active among the tested phenolic compounds.

Cholesterol concentration and fatty acid profile of red deer (Cervus elaphus) meat

The effects of gender and age on intramuscular fat (IMF) levels, cholesterol concentration, and fatty acid composition were investigated in the semitendinosus (ST) and triceps brachii (TB) muscles of feral red deer (Cervus elaphus). Six stags of >2years of age, four hinds of 1year, and six calves of 6months were shot in Slovenia. Generally, all parameters measured were influenced by interaction of muscle and treatment group (hinds, stags and calves) at the 5% level or less. In ST muscle, the IMF levels were highest for hinds. In the TB muscle, cholesterol was lower for stags than for hinds and calves. The saturated fatty acids were the highest for stags and the mono-unsaturated fatty acids for hinds. The polyunsaturated fatty acids (PUFAs) were the highest for calves and lowest for hinds. The n-3 PUFAs were the lowest for hinds. In both muscles, the calves had higher n-6 PUFAs than stags and hinds. Only the ST muscle of the hinds contained >1% (1.44%) of the conjugated linoleic acid isomer 18:2cis-9,trans-11, while in the TB of hinds and calves this fatty acid was higher than with stags. We conclude that gender and age of feral red deer influence the IMF content, the cholesterol concentration, and the fatty acid composition of the meat.

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

BackgroundFK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses.ResultsThree putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively.ConclusionsOur results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Construction of a New Class of Tetracycline Lead Structures with Potent Antibacterial Activity through Biosynthetic Engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Fractionation of Phenolic Compounds Extracted from Propolis and Their Activity in the Yeast Saccharomyces cerevisiae

We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds.

Phenolic content of strawberry spreads during processing and storage.

This study provides a comprehensive and systematic evaluation of phenolics in strawberry spreads processed according to different industrial procedures and stored under several storage conditions for up to 19 weeks. Total phenolics were determined spectrophotometrically, and individual phenolics were determined by combined liquid chromatography and mass spectrometry: six anthocyanins, four phenolic acids, two flavonols, one flavanol, and one flavone. During storage, the phenolics were modified. The total anthocyanins, vanillic acid, kaempferol, and luteolin decreased, while salicylic and gallic acids increased. Total phenolics, cyanidin 3-(6″-succinyl-glucoside) (here observed for the first time), protocatechuic acid, quercetin, and catechin remained stable. The best phenolic retention was observed in spreads stored at 4 °C. Therefore, the proposed storage process (use of a cold chain) indicates good retention of phenolics in strawberry spreads, which maintain high nutritional and sensorial quality.

Stability and transformation of products formed from dimeric dehydroascorbic acid at low pH

Dehydroascorbic acid dimer (DDHA) is the commercially available form of oxidised vitaminC. When DDHA enters an aqueous environment, it will interact with water, but the details of this process are not well defined. Hydration of DDHA was analyzed as a function of concentration, pH and temperature. Above pH 5 only hydrated dehydroascorbic acid monomer (DHA) and its degradation product 2,3-diketogulonic acid (DKG) were observed. At pH lower than4 and in MilliQ water, relatively stable hydrated dimeric species were additionally detected. They are intermediates formed from DDHA and transformed into DKG, either directly or through DHA. A lower temperature of dissolution favours the formation of hydrated dimeric products, while the concentration of DDHA has no effect on their yield. All products were separated chromatographically and analyzed by three detectors: diode array, refractive index and electrospray ionisation mass spectrometry. The stability and transformations of products were studied in the mixture and in the isolated fractions. Two initially formed monohydrated products are transformed to a dihydrated product that is degraded to DHA and DKG. Once formed, the dihydrated product and one of the monohydrated products are sufficiently stable to be identified at neutral pH. All three hydrated products are interconvertible and can be also formed after dissolution of dried DHA.

LC–MS analysis of phenolic compounds and antioxidant activity of buckwheat at different stages of malting

The impact of malting on the profile of the phenolic compounds and the antioxidant properties of two buckwheat varieties was investigated. The highest relative increases in phenolic compounds were observed for isoorientin, orientin, and isovitexin, which are consequently major inducible phenolic compounds during malting. Only a minor relative increase was observed for the most abundant phenolic compound, rutin. The radical-scavenging activity of buckwheat seeds was evaluated using ABTS and DPPH assays. A considerable increase in total phenolic compounds and higher antioxidant activity were observed after 64h of germination, whereas kilning resulted in decreased total phenolic compounds and antioxidant activity. Higher antioxidant activities for extracts were found for buffered solvents than for pure methanol and water. Changes in the composition of the phenolic compounds and increased antioxidant content were confirmed by several methods, indicating that buckwheat malt can be used as a food rich in antioxidants.

Effects of industrial and home-made spread processing on bilberry phenolics.

Bilberries processed into spreads represent an important source of anthocyanins if these remain rich in the final product. The effects of thermal processing were studied with non-ground and ground bilberries processed into spreads according to industrial and home-made procedures. Samples were analysed by LC-DAD-MS/MS and LC-MS. The spreads had 28-60% less total phenolics, 4-62% less anthocyanins, and 1-fold to 2-fold more phenolic acids and total flavonols than the bilberries, but approximately equal flavanols. The home-made spread from ground bilberries had ca. 26% higher antioxidant activity. Delphinidin 3-glucoside and cyanidin 3-glucoside were taken through the two spread procedures, with their degradation to gallic acid (38-57%), protocatechuic acid (1-2%) and 2,4,6-trihydroxybenzaldehyde determined. The amounts of gallic and protocatechuic acids did not reflect well for anthocyanin degradation. The industrial spread procedure with non-ground bilberries is a more suitable procedure to maintain the final content of anthocyanins.

Effect of heat treatment of camelina (Camelina sativa) seeds on the antioxidant potential of their extracts.

The effect of different heat treatments of camelina (Camelina sativa) seeds on the phenolic profile and antioxidant activity of their hydrolyzed extracts was investigated. The results showed that total phenol contents increased in thermally treated seeds. Heat treatment affected also the quantities of individual phenolic compounds in extracts. Phenolics in unheated camelina seeds existed in bound rather than in free form. A temperature of 160 °C was required for release of insoluble bound phenolics, whereas lower temperatures were found to be optimal to liberate those present as soluble conjugates. The best reducing power and alkyl peroxyl radical scavenging activity in the emulsion was expressed by phenolics which were bound to the cell wall, whereas the best iron chelators and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavengers were found to be those present in free form. The heat treatment of seeds up to 120 °C increased the reducing power and DPPH• radical scavenging ability of extracts, but negatively affected iron chelating ability and their activity in an emulsion against alkyl peroxyl radicals.