Phenolic Profile, Antioxidant Activity and Amino Acid Composition of Moringa Leaves Fermented with Edible Fungal Strains.

Solid-state fermentation (SSF) is widely recognised as a technique to increase the bioactive potential and nutritional value of plant materials. However, the effect of this biotreatment differs for individual substrates. This study aimed to evaluate the impact of SSF with filamentous fungi (Rhizopus, Aspergillus, and Neurospora) on a moringa leaf phenolic profile, antioxidant activity, and amino acid composition. A total of 43 phenolic compounds were determined in the dried leaves analysed by HPLC-ESI-TOF-MS. The leaves contained 11.79 mg/g of free phenolics: flavonols (80.6%, mainly quercetin and kaempferol glycosides), hydroxycinnamic acid derivatives (12.3%), vitexin and vicenin (6.9%), and a small amount of lignan (isolariciresinol isomers). The result of the 1-day fermentation was a slight enhancement in the concentration of individual free phenolics (flavones) and the antioxidant activity of the leaves. However, extending the incubation period caused a significant decrease in those parameters and cannot be recommended for obtaining a food fortificant from moringa leaves. In contrast, the 3-day fermentation with N. intermedia led to a 26% average accumulation of individual amino acids. Therefore, the SSF with Neurospora can be a promising method for improving the nutritional composition of moringa leaves and needs further investigation.

Antioxidant and Antimicrobial Activity of Hydroethanolic Leaf Extracts from Six Mediterranean Olive Cultivars.

Phenolic profiles, antioxidant, and antimicrobial activities of hydroethanolic olive leaf extracts from six Mediterranean olive cultivars (Croatian: Lastovka, Levantinka, Oblica; Italian: Moraiolo, Frantoio, Nostrana di Brisighella) were investigated. As expected, various distributions of phenolic levels were observed for each cultivar and the total phenolic content showed high variability (ranging from 4 to 22 mg GAE/g of dry extract), with the highest amount of phenolics found in the Oblica sample, which also provided the highest antiradical (ORAC) and reducing activity (FRAP). The screening of individual compounds was performed by HPLC-PDA-ESI-QTOF-MS and the main detected compounds were oleuropein, hydroxytyrosol, oleoside/secologanoside, verbascoside, rutin, luteolin glucoside, hydroxyoleuropein, and ligstroside. While the antioxidant activity of the samples was relatively high, they showed no bactericidal and bacteriostatic activity against E. coli and S. Typhimurium; weak activity against Staphylococcus aureus, Bacillus cereus, and Listeria innocua; and inhibitory effects against Campylobacter jejuni at 0.5 mg dry extract/mL. The obtained results support the fact that olive leaf extracts, and especially those from the Oblica cultivar, could potentially be applied in various industries as natural preservatives and effective and inexpensive sources of valuable antioxidants.

Solid State Fermentation of Olive Leaves as a Promising Technology to Obtain Hydroxytyrosol and Elenolic Acid Derivatives Enriched Extracts.

Extraction of valuable bioactive compounds from olive leaves is a hot topic and the use of sustainable and green technologies is mandatory in terms of circular economy. In this way, the use of fermentation technologies showed very interesting results in terms of phenolic compound recovery. Because of that in this work the use of solid state fermentations, as valuable tool to improve the phenolic extraction has been checked. Aspergillus oryzae (in mycelium and spore form), Aspergillus awamori and Aspergillus niger were used as fermentation microrganisms. Phenolic compounds were determined by HPLC-ESI-TOF-MS and, to our knowledge, new compounds have been tentatively identified in olive leaves. Fermentation using mycelium of Aspergillus awamori, Aspergillus niger and Aspergillus oryzae were effective to increase both hydroxytyrosol and elenolic acid derivatives whereas the use of spores of Aspergillus oryzae caused a loss of hydroxytyrosoyl derivatives, contrary the content of elenolic derivatives are comparable with the other fermentation treatments and higher than control. The proposed fermentation processes using the mycelium of Aspergillus awamori, Aspergillus niger and Aspergillus oryzae lead to an increase the hydroxytyrosyl and elenolic acid derivatives and could be used at industrial scale to obtain enriched extracts.

Evaluating the reliability of specific and global methods to assess the phenolic content of virgin olive oil: Do they drive to equivalent results?

Despite the huge number of different published methodologies, there is an open debate regarding which one is the most convenient analytical strategy for the determination of phenolic compounds from virgin olive oils. Diverse technical issues together with the disparity of criteria regarding results expression cause a lot of confusion. Herewith, a systematic comparison between specific (a powerful and LC-MS method) and global methodologies (the Folin-Ciocalteau (FC) colorimetric assay, the International Olive Council (IOC) method and hydrolysis plus HPLC-DAD) has been carried out. Thus, these strategies have been applied to the analysis of 50 extra virgin olive oils (covering all the possible quantitative ranges of these substances). This is the first time in which the individual LC-MS quantification of so many phenolic substances is included in this kind of comprehensive comparison. The outcomes of all the strategies have been thoroughly confronted and their equivalence (or divergence) has been carefully evaluated, establishing possible correspondence factors. The LC-MS individual determination with the pure standard of every analyte represented the ideal situation; when only the commercially available standards were used, a drastic change was observed in the absolute concentrations of oleuropein derivatives (in terms of hydroxytyrosol). Total phenolic content (summing individual levels) proved to be higher (1.9-3.0 times when data was expressed in mg/kg) than the values given by the three non-specific methods (with R2 from 0.84 to 0.90). In any case, the IOC method, the FC assay and the hydrolysis approach could be considered as feasible strategies when a global value is pursued. Good correlations between their results were found (R2 > 0.89), with the following equivalence factors: FC(mg caffeic acid/kg) ≈ 0.60 IOC(mg TY/kg); IOC(mg TY/kg) ≈ 1.27 Sum acid hydrolysis(mg TY+HTY/kg); FC(mg HTY/kg) ≈ 1.04 Sum acid hydrolysis(mg TY+HTY/kg).

High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization.

We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

The ß-sliding clamp directs the localization of HdaA to the replisome in Caulobacter crescentus.

The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the ß-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.

Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.