Multifunctional Pomegranate Peel Microparticles with Health-Promoting Effects for the Sustainable Development of Novel Nutraceuticals and Pharmaceuticals.

Recovering the bioactive components from pomegranate peel (PP) in the fruit-processing industry has attracted great attention in terms of minimizing the waste burden, as well as providing a new source of a multitude of functional compounds. The present study aimed to develop a feasible microencapsulation process of PP extract by using pectin and a pectin/2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) blend as coating materials. Microsized powders obtained by a spray drying technique were examined in terms of technological characteristics, exhibiting high powder yield and desirable moisture content, flowability, and cohesive properties. Assuming that the interactions with the used biopolymers occur on the surface hydrophobic domain, their presence significantly improved the thermal stability of the microencapsulated powders up to 200 °C. The health-promoting effects of PP have been associated with its high content in ellagitannins, particularly punicalagin. The obtained PP powders exhibited strong antioxidant and hypoglycemic potential, while an antimicrobial assay revealed their potent activity against Gram-positive bacteria. Additionally, an in vitro release study suggested that the used biopolymers can modify the release of target bioactive compounds, thus establishing a basis for developing an oral-controlled release system. Altogether, biowaste valorization from PP by the production of effective multifunctional microsized powders represents a sustainable way to obtain novel nutraceuticals and/or pharmaceuticals.

Microencapsulated Bilberry and Chokeberry Leaf Extracts with Potential Health Benefits.

The aim of the research was to develop microencapsulated powders of bilberry and chokeberry extracts via the spray drying technique. Two biopolymers, pectin alone and in combination with HP-ß-CD, were used to preserve the antioxidant, hypoglycemic, photoprotective, and antimicrobial bioactivity of the berry leaf extracts. Moreover, the formed powders were characterized in terms of technological, chemical, and several biological properties. The obtained micro-sized powders (mean average particle diameter from 3.83 to 5.94 µm) demonstrated a process yield of up to 73%. The added biopolymers improved the flowability and cohesive properties of the powders and increased their thermal stability to 170 °C. The total content of polyphenolics in the powders ranged from 323.35 to 367.76 mg GAE/g DW for bilberry and from 186.85 to 227.59 mg GAE/g DW for chokeberry powders; meanwhile, chlorogenic acid was the predominant compound in powders. All samples showed stronger α-glucosidase inhibitory activity (IC50 values ranged from 5.00 to 19.59 µg/mL) compared with the reference standard. The study confirmed that spray drying is a suitable method for the preservation of the polyphenolic-rich extracts, while the addition of carriers has a positive effect on the improvement of microencapsulated powders' properties.

Insights into Endophytic and Rhizospheric Bacteria of Five Sugar Beet Hybrids in Terms of Their Diversity, Plant-Growth Promoting, and Biocontrol Properties.

Sugar beet is the most important crop for sugar production in temperate zones. The plant microbiome is considered an important factor in crop productivity and health. Here, we investigated the bacterial diversity of seeds, roots, and rhizosphere of five sugar beet hybrids named Eduarda (ED), Koala (KO), Tibor (T), Tajfun (TF), and Cercospora-resistant (C). A culture-independent next-generation sequencing approach was used for the further investigation of seed-borne endophytes. Hybrid-associated bacteria were evaluated for their plant growth-promoting (PGP) characteristics, antagonistic activity towards Cercospora beticola and several Fusarium strains in dual culture assays, and drought and salinity tolerance. High-throughput sequencing revealed that the Proteobacteria phylum was most dominant in the seeds of all hybrids, followed by Cyanobacteria and Actinobacteriota. The predominant genus in all hybrids was Pantoea, followed by Pseudomonas, Acinetobacter, Chalicogloea, Corynebacterium, Enterobacter, Enterococcus, Glutamicibacter, Kosakonia, and Marinilactibacillus. Unique genera in the hybrids were Pleurocapsa and Arthrobacter (T), Klebsiella (TF), Apibacter (ED), and Alloscardovia (KO). The genera that were most represented in one hybrid were Weissella and Staphylococcus (TF); Streptococcus (T); Gardnerella, Prevotella, and Rothia (KO); and Gilliamella, Lactobacillus, and Snodgrassella (ED). Thirty-two bacteria out of 156 isolates from the rhizosphere, roots, and seeds were selected with respect to various plant growth-promoting activities in vitro, i.e., nitrogen fixation, phosphate solubilization, siderophore production, indole-3-acetic acid production, 1-aminocyclopropane-1-carboxylic acid deaminase activity, hydrogen cyanide production, exoenzymatic activity (amylase, protease, lipase, cellulase, xylanase, mannanases, gelatinase, and pectinase), mitigation of environmental stresses, and antifungal activity. Mixta theicola KO3-44, Providencia vermicola ED3-10, Curtobacterium pusillum ED2-6, and Bacillus subtilis KO3-18 had the highest potential to promote plant growth due to their multiple abilities (nitrogen fixation, phosphate solubilization, production of siderophores, and IAA). The best antagonistic activity towards phytopathogenic fungi was found for Bacillus velezensis C3-19, Paenibacillus polymyxa C3-36 and Bacillus halotolerans C3-16/2.1. Only four isolates B. velezensis T2-23, B. subtilis T3-4, B. velezensis ED2-2, and Bacillus halotolerans C3-16/2.1 all showed enzymatic activity, with the exception of xylanase production. B. halotolerans C3-16/2.1 exhibited the greatest tolerance to salinity, while two B. subtilis strains (C3-62 and TF2-1) grew successfully at the maximum concentration of PEG. The current study demonstrates that sugar beet-associated bacteria have a wide range of beneficial traits and are therefore highly promising for the formulation of biological control and PGP agents.

Culture-Dependent and Metabarcoding Characterization of the Sugar Beet (Beta vulgaris L.) Microbiome for High-Yield Isolation of Bacteria with Plant Growth-Promoting Traits.

The diversity of plant-associated bacteria is vast and can be determined by 16S rRNA gene metabarcoding. Fewer of them have plant-beneficial properties. To harness their benefits for plants, we must isolate them. This study aimed to check whether 16S rRNA gene metabarcoding has predictive power in identifying the majority of known bacteria with plant-beneficial traits that can be isolated from the sugar beet (Beta vulgaris L.) microbiome. Rhizosphere and phyllosphere samples collected during one season at different stages of plant development were analyzed. Bacteria were isolated on rich unselective media and plant-based media enriched with sugar beet leaves or rhizosphere extracts. The isolates were identified by sequencing the 16S rRNA gene and tested in vitro for their plant-beneficial properties (stimulation of germination; exopolysaccharide, siderophore, and HCN production; phosphate solubilization; and activity against sugar beet pathogens). The highest number of co-occurring beneficial traits was eight, found in isolates of five species: Acinetobacter calcoaceticus, Bacillus australimaris, B. pumilus, Enterobacter ludwiigi, and Pantoea ananatis. These species were not detected by metabarcoding and have not previously been described as plant-beneficial inhabitants of sugar beets. Thus, our findings point out the necessity of a culture-dependent microbiome analysis and advocate for low-nutrient plant-based media for high-yield isolation of plant-beneficial taxa with multiple beneficial traits. A culture-dependent and -independent approach is required for community diversity assessment. Still, isolation on plant-based media is the best approach to select isolates for potential use as biofertilizers and biopesticides in sugar beet cultivation.

Bioassay-Guided Skin-Beneficial Effects of Fractionated Sideritis raeseri subsp. raeseri Extract.

This study aimed to evaluate the potentials for skin-beneficial effects of the hydroethanolic extract of the aerial parts of ironwort (Sideritis raeseri Boiss. & Heldr. subsp. raeseri) and its fractions (petroleum ether, ethyl acetate, n-butanol and water). For these purposes, chemical analysis and in vitro antioxidant, anti-tyrosinase and antimicrobial assays, as well as determination of sun protection factor (SPF), were performed. The highest total phenolic content and quantity of individual flavonoids, chlorogenic acid and verbascoside were determined in the n-butanol fraction, which is in line with results obtained for antioxidant activity. The greatest antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the most common causative agents of the skin infections in humans, was exhibited by the ethyl acetate fraction. The strongest anti-tyrosinase activity was shown by the hydroethanolic extract (52.64% of inhibition at 100 µg/mL). Almost all tested samples showed photoprotective activity with SPF higher than 6 obtained at a low concentration of 0.1 mg/mL, which was more than 15 for the butanol fraction. These findings revealed that the hydroethanolic extract of S. raeseri aerial parts could be a valuable ingredient in the formulation of cosmetic and medicinal products intended to overcome various skin disorders.

Ultrasound-Assisted Natural Deep Eutectic Solvents Extraction of Bilberry Anthocyanins: Optimization, Bioactivities, and Storage Stability.

Bilberry fruits (Vaccinium myrtillus L.) are one of the richest natural sources of anthocyanins and are widely used due to their pharmacological and nutritional properties. To ensure their maximum application potential, it is necessary to overcome the limitations of conventional extraction solvents and techniques. This study aimed to develop a green method for bilberry anthocyanin extraction using natural deep eutectic solvents (NaDES) integrated with ultrasound-assisted extraction (UAE) in order to define extraction conditions that will prevent decomposition of the anthocyanins or the loss of bioactivity. After a screening of ten different NaDES, choline chloride:sorbitol (1:1) was selected as the most effective. Furthermore, the influence analysis and optimization of the NaDES-UAE extraction conditions were carried out employing response surface methodology. The optimal conditions were found to be an extraction time of 37.63 min, a temperature of 48.38 °C, and 34.79% (w/w) water in NaDES. The extraction yields of target compounds under optimized extraction conditions were 0.27 mg/g DW of cyanidin-3-O-glucoside and 2.12 mg CGE/g DW of TAC. The obtained optimized extract showed promising radical scavenging and antimicrobial activity. A stability study with the optimized extract revealed that refrigerated storage at 4 °C in the dark provided the best anthocyanins preservation. Overall, the developed NaDES-UAE method showed promising application potential and can be considered as a high-efficiency green alternative to conventional anthocyanins extraction methods, enabling the preservation of active ingredients and the bioactivity of extracts.

The Microbiome of the 'Williams' Pear Variety Grown in the Organic Orchard and Antifungal Activity by the Autochthonous Bacterial and Yeast Isolates.

The total diversity of bacterial and fungal communities associated with the phyllosphere (fruits and leaves) of the 'Williams' pear variety was analyzed in two phenological stages during fruit development and maturation. The antagonistic potential of autochthonous bacterial and yeast isolates against phytopathogenic fungi was also evaluated. A metabarcoding approach revealed Pantoea, Sphingomonas, Hymenobacter, Massilia, and Pseudomonas as dominant bacterial constituents of the pear phyllosphere, whilst most abundant among the fungal representatives identified were Metschnikowia, Filobasidium, Aureobasidiumpullulans, Botrytis cinerea, and Taphrina. The traditional culturable approach revealed that the Pseudomonas genus with P. graminis, P. putida, and P. congelans was most prevalent. The most frequently cultivated fungal representatives belonged to the genus Fusarium with six identified species. A broad range of the antagonistic activity was detected for the Hannaella luteola and Metschnikowia pulcherrima yeasts, significantly affecting the growth of many fungal isolates in the range of 53-70%. Fusarium sporotrichioides was the most susceptible fungal isolate. The autochthonous antagonistic yeasts H. luteola and M. pulcherrima might be powerful biological control agents of postharvest diseases caused by Fusarium spp. and common pathogens like Monilinia laxa, Botrytis cinerea, Alternaria tenuissima, and Cladosporium cladosporioides.

Chemical Defence in a Millipede: Evaluation and Characterization of Antimicrobial Activity of the Defensive Secretion from Pachyiulus hungaricus (Karsch, 1881) (Diplopoda, Julida, Julidae).

The chemical defence of the millipede Pachyiulus hungaricus is reported in the present paper, in which a chemical characterization is given and antimicrobial activity is determined. In total, independently of sex, 44 compounds were identified. All compounds belong to two groups: quinones and pentyl and hexyl esters of long-chain fatty acids. The relative abundances of quinones and non-quinones were 94.7% vs. 5.3% (males) and 87.3% vs. 12.7% (females), respectively. The two dominant quinones in both sexes were 2-methyl-1,4,-benzoquinone and 2-methoxy-3-methyl-1,4-benzoquinone. Antibacterial and antifungal activity of the defensive secretion was evaluated in vitro against seven bacterial strains and eight fungal species. With the aid of a dilution technique, the antimicrobial potential of the secretion and high sensitivity of all tested strains were confirmed. The lowest minimum concentrations of these compounds (0.20-0.25 mg/mL) were sufficient for inhibition of Aeromonas hydrophila, Listeria monocytogenes and Methicillin resistant Staphylococcus aureus (MRSA). The growth of eight tested fungal species was inhibited by slightly lower concentrations of the secretion, with Fusarium equiseti as the most sensitive fungus and Aspergillus flavus as the most resistant. Values of MIC and MFC in the employed microdilution assay ranged from 0.10 to above 0.35 mg/mL. The given extract contains antimicrobial components potentially useful as therapeutic agents in the pharmaceutical and agricultural industries.

Metabolic changes in Euphorbia palusrtis latex after fungal infection.

The variations of metabolic profile of the latex of wild-growing Euphorbia palustris was carried out using multivariate analysis of 1H NMR spectral data. One population was infected with fungi Fusarium sporotrichioides, Fusarium proliferatum and Alternaria alternata, while the other consisted of healthy plant species. The non-polar metabolites of latex extracts such as benzoyl ingenol-laurate, amyrin decadienoate esters, cis-1,4-polyisoprene, and 24-methylenecycloartanol were identified using 1H and 2D NMR spectra. Principal component analysis of 1H NMR data provided a clear discrimination between the latex of infected and healthy plants. Minimum inhibitory concentration and minimum fungicidal concentration values of the latex extracts of healthy and infected plants were determined. The latex of infected plants was found to contain higher levels of benzoyl ingenol-laurate and 24-methylenecycloartanol, of which concentrations were strongly correlated with the antifungal activities of the latex.

Essential oil from black currant buds as chemotaxonomy marker and antimicrobial agent.

Dormant buds are recognized as valuable side product of the blackcurrant cultivation. Four blackcurrant varieties cultivated in Serbia, i.e., Ben Sarek, Ometa, Ben Lomond, and Ben Nevis, were evaluated for the content, chemical composition, and antimicrobial activity of their bud essential oils. The oil yields of buds harvested during two different growth periods ranged from 1.2-2.0%, and the variety Ometa had the highest yield among the tested varieties. GC-FID and GC/MS analysis of the oils allowed the identification of eight main components, i.e., α-pinene (1.6-5.4%), sabinene (1.9-38.4%), δ-car-3-ene (13.0-50.7%), ß-phellandrene (2.9-18.0%), terpinolene (6.6-11.9%), terpinen-4-ol (0.9-6.6%), ß-caryophyllene (3.8-10.4%), and α-humulene (0.2-4.1%). In addition, the similarity degree of the essential-oil compositions of buds harvested from the upper and lower parts of the shrubs was investigated by hierarchical clustering. All essential oils originating from the same genotype were grouped in the same cluster, indicating the reliability of essential oils as chemotaxonomic markers. For more detailed chemotaxonomic investigations, the three compounds with the greatest variance were chosen, i.e., sabinene, δ-car-3-ene, and ß-phellandrene, which proved to be efficient for the variety distinction. Factor analysis showed that the essential-oil composition as chemotaxonomic marker in blackcurrants was more reliable for variety Ben Sarek than for variety Ben Nevis. Moreover, it was demonstrated that the essential oils had very strong inhibitory activity against all tested microorganisms. Fungi were more sensitive than bacteria; indeed their growth was completely inhibited at much lower concentrations. In comparison to commercial antibiotics, significantly lower concentrations of the oils were necessary for the complete inhibition of fungal growth.

Antioxidant, cytotoxic, and antimicrobial activity of Alnus incana (L.) ssp. incana Moench and A. viridis (Chaix) DC ssp. viridis extracts.

Antioxidant, cytotoxic, and antimicrobial activities of leaves, bark, and cone extracts of Alnus incana (L.) Moench ssp. incana and endemic species A. viridis (Chaix) DC ssp. viridis were evaluated. All extracts were found to be strong 1,1-diphenyl-2-picrylhydrazyl free radical scavengers, exhibiting 50% inhibitory concentration (IC(50)) values of 3.3-18.9 microg/mL, and also showed activity in inhibition of lipid peroxidation with IC(50) values ranging from 38.5 to 157.4 microg/mL. A. incana and A. viridis extracts exhibited significant cytotoxic effects toward HeLa cells, with IC(50) values ranging from 26.02 to 68.5 microg/mL. The most active extract of A. incana bark also contained great amounts of total phenolics (316.2 mg of gallic acid equivalents/g). In our experiment all extracts were virtually nontoxic on brine shrimps. Extracts were screened for activity against 15 microorganisms, and all extracts investigated showed antimicrobial activity. The most active were dry extracts of cones of A. incana and A. viridis with minimum inhibitory concentration values ranging from 0.117 to 0.129 mg/mL.

Antimicrobial activity of Gentiana lutea L. extracts.

Methanolic extracts of flowers and leaves of Gentiana lutea L., together with the isolated compounds mangiferin, isogentisin and gentiopicrin, were used to investigate the antimicrobial activity of the plant. A variety of Gram-positive and Gram-negative bacteria as well as the yeast Candida albicans has been included in this study. Both extracts and isolated compounds showed antimicrobial activity with MIC values ranging from 0.12-0.31 mg/ml. Our study indicated that the synergistic activity of the pure compounds may be responsible for the good antimicrobial effect of the extracts. Quantification of the secondary metabolites was performed using HPLC.

Phenolic content and radical scavenging capacity of berries and related jams from certificated area in Serbia.

Chemical composition and the effects of domestic processing and storage were studied in organically cultivated or wild growing berries, harvested in certificated area in Serbia. The total phenolics content in fresh berries ranged from 380 to 1660 mg GAE/100 g. The phenolic content decreased during the processing to jams, and storage in almost all samples, except in black currant. Among fresh berries, the highest amount of total anthocyanins was found in bilberry (0.61 +/- 0.03 %). Processing and storage caused decrease in anthocyanin content. The most ubiquitous anthocyanin aglycone was found to be cyanidin. Ellagic acid was detected in fresh and frozen black raspberries and related jams, and its content increases during thermal processing to jam. Among fresh berries, bilberry showed the strongest DPPH radical scavenging activity with an IC(50) value of 0.82 mg/ml. However, frozen as well as processed berries in our study still showed comparable high values of radical scavenging activity. After 9 months of storage all investigated jams were microbiologicaly correct. Regardless of the losses in the amount of total phenolics, total anthocyanins and radical scavenging activity, frozen berries and related jams are still excellent sources of nutritional substances with antioxidant potential.