ICP-MS Determination of Antimicrobial Metals in Microcapsules.

Silver (Ag) and zinc (Zn) are very powerful antimicrobial metals. Therefore, in this research, a high-throughput, sensitive, and rapid method was developed for the determination of Ag and Zn in microcapsules using inductively coupled plasma mass spectrometry (ICP-MS). The sample preparation procedure employed simple microwave digestion of the microcapsules with 55.55% v/v HNO3 and 44.45% v/v H2O2. The method was applied to determine Ag and Zn in microcapsule samples of different sizes (120 and 450 µm) after their preparation with and without chitosan. Prepared microcapsules, after characterization, were bonded to a polymer carrier by sol-gel procedure and the materials were characterized by FTIR spectroscopy and high-resolution optical microscopy. Significant differences were found in Ag and Zn levels between microcapsules samples prepared with and without chitosan. The results have shown that samples with chitosan had up to 20% higher levels of Zn than Ag: 120 µm microcapsules contained 351.50 µg/g of Ag and 85.51 µg/g of Zn, respectively. In contrast, samples prepared without chitosan showed larger overall variability: In microcapsules with a diameter of 120 µm, the amounts of antimicrobial metals were 98.32 µg/g of Ag and 106.75 µg of Zn, respectively. Moreover, 450 µm microcapsules contained 190.98 µg/g of Ag and 121.35 µg/g of Zn. Those quantities are high enough for efficient antimicrobial activity of newly prepared microcapsules, enabling the application of microcapsules in different antimicrobial coatings.

Synthesis, Characterization, and Encapsulation of Novel Plant Growth Regulators (PGRs) in Biopolymer Matrices.

Novel plant growth regulators (PGRs) based on the derivatives of dehydroamino acids 2,3-dehydroaspartic acid dimethyl ester (PGR1), Z-isomer of the potassium salt of 2-amino-3-methoxycarbonylacrylic acid (PGR2) and 1-methyl-3-methylamino-maleimide (PGR3) have been synthesized and their growth-regulating properties investigated. Laboratory testing revealed their plant growth-regulating activity. PGR1 showing the most stimulating activity on all laboratory tested cultures were used in field experiments. Results showed that PGR1 is a highly effective environmentally friendly plant growth regulator with effects on different crops. Biopolymeric microcapsule formulations (chitosan/alginate microcapsule loaded with PGR) suitable for application in agriculture were prepared and characterized. Physicochemical properties and release profiles of PGRs from microcapsule formulations depend on the molecular interactions between microcapsule constituents including mainly electrostatic interactions and hydrogen bonds. The differences in the microcapsule formulations structure did not affect the mechanism of PGRs release which was identified as diffusion through microcapsules. The obtained results opened a perspective for the future use of microcapsule formulations as new promising agroformulations with a sustained and target release for plant growth regulation.

Encapsulation of Synthesized Plant Growth Regulator Based on Copper(II) Complex in Chitosan/Alginate Microcapsules.

A new copper complex, trans-diaqua-trans-bis [1-hydroxy-1,2-di (methoxycarbonyl) ethenato] copper (abbreviation Cu(II) complex), was synthesized and its plant growth regulation properties were investigated. The results show a sharp dependence of growth regulation activity of the Cu(II) complex on the type of culture and its concentration. New plant growth regulator accelerated the development of the corn root system (the increase in both length and weight) but showed a smaller effect on the development of the wheat and barley root systems. Stimulation of corn growth decreased with increasing Cu(II) complex concentration from 0.0001% to 0.01% (inhibition at high concentrations-0.01%). The development of corn stems was also accelerated but to a lesser extent. Chitosan-coated calcium alginate microcapsules suitable for delivery of Cu(II) complex to plants were prepared and characterized. Analysis of the FTIR spectrum showed that complex molecular interactions between functional groups of microcapsule constituents include mainly electrostatic interactions and hydrogen bonds. Microcapsules surface exhibits a soft granular surface structure with substructures consisting of abundant smaller particles with reduced surface roughness. Release profile analysis showed Fickian diffusion is the rate-controlling mechanism of Cu(II) complex releasing. The obtained results give new insights into the complexity of the interaction between the Cu(II) complex and microcapsule formulation constituents, which can be of great help in accelerating product development for the application in agriculture.

Design and synthesis of novel antimicrobial peptide scaffolds.

Muramic acid (Mur), a sugar amino acid (SAA), is present in the cell walls of bacteria asN-acetyl muramic acid (MurNAc) where together with ofN-acetylglucosamine (GlcNAc) and peptide makes main building block of peptidoglycan (PGN). It was challenging to incorporate muramic acid as SAA characteristic for bacteria into the peptides and investigate the antimicrobial activity of these scaffolds. Four building units were used in designing the desired peptide: muramic acid, tetrapeptide Leu-Ser-Lys-Leu, Nε-Lys, and Asn. Positions of three components were changeable while the position of Asn was always C-terminal (in linear peptides). The glycopeptide libraries of linear and cyclic peptides were synthesized using solid-phase peptide synthesis (SPPS). The antimicrobial effect of linear and cyclic glycopeptides, as well as the LSKL sequence used as a control, was investigated on several standard laboratory microbial strains. Liner glycopeptide with sequences Leu-Ser-Lys-Leu-Nε-Lys-Mur-Asn was active onStaphylococcus aureus(Gram-positive bacteria). Prepared compounds did not show activity towards applied tumor and normal human cell lines.

C-Linked Glycomimetic Libraries Accessed by the Passerini Reaction.

Carbohydrates and their conjugates are the most abundant natural products, with diverse and highly important biological roles. Synthetic glycoconjugates are versatile tools used to probe biological systems and interfere with them. In an endeavor to provide an efficient route to glycomimetics comprising structurally diverse carbohydrate units, we describe herein a robust, stereoselective, multicomponent approach. Isopropylidene-protected carbohydrate-derived aldehydes and ketones were utilized in the Passerini reaction, giving different glycosylated structures in high yields and diastereoselectivities up to 90:10 diastereomeric ratio (d.r). Access to highly valuable building blocks based on α-hydroxy C-glycosyl acids or more complex systems was elaborated by simple post-condensation methodologies.

Synthesis of Glycomimetics by Diastereoselective Passerini Reaction.

We describe the utilization of bis-isopropylidene-protected d-fructose-derived aldehyde in the Passerini reaction with various acids and isocyanides. A library of densely functionalized glycomimetics bearing up to 3 carbohydrate units was obtained in high yields and diastereoselectivities. The configuration of the newly formed stereocenter was determined and the diastereoselectivity was rationalized by DFT calculations.

α-Hydrazino Acid Insertion Governs Peptide Organization in Solution by Local Structure Ordering.

In this work, we have applied the concept of α-hydrazino acid insertion in a peptide sequence as a means of structurally organizing a potential protein-protein interactions (PPI) inhibitor. Hydrazino peptides characterized by the incorporation of an α-hydrazino acid at specific positions introduce an additional nitrogen atom into their backbone. This modification leads to a change in the electrostatic properties of the peptide and induces the restructuring of its hydrogen bonding network, resulting in conformational changes toward more stable structural motifs. Despite the successful use of synthetic hydrazino oligomers in binding to nucleic acids, the structural changes due to the incorporation of α-hydrazino acid into short natural peptides in solution are still poorly understood. Based on NMR data, we report structural models of p53-derived hydrazino peptides with elements of localized peptide structuring in the form of an α-, ß-, or γ-turn as a result of hydrazino modification in the peptide backbone. The modifications could potentially lead to the preorganization of a helical secondary peptide structure in a solution that is favorable for binding to a biological receptor. Spectroscopically, we observed that the ensemble averaged rapidly interconverting conformations, including isomerization of the E-Z hydrazide bond. This further increases the adaptability by expanding the conformational space of hydrazine peptides as potential protein-protein interaction antagonists.

Determination of Bioactive Components in Mandarin Fruits: A Review.

During the last decade, there has been a continuous rise in the consumption of fresh easy-to-peel mandarins. However, the majority of the knowledge comes from other citrus fruit, like orange, while there are relatively few studies about mandarins and no comprehensive research on literature data about them. One of the most important steps in the analytical process is sample preparation. Its value is evident in analyzing the samples with complex matrices, such as in mandarin fruit. In addition, mandarin contains hundreds to thousands of various compounds and metabolites, some of them present in extremely low concentrations, that interfere with the detection of one another. Hence, mandarin samples are commonly pretreated by extraction to facilitate analysis of bioactive compounds, improve accuracy and quantification levels. There is an abundance of extraction techniques available, depending on the group of compounds of interest. Finally, modern analytical techniques, have been applied to cope with numerous bioactive compounds in mandarins. Considering all the above, this review aims to (i) list the most valuable procedures of sample preparation, (ii) highlight the most important techniques for extraction of bioactive compounds from mandarin fruit, and (iii) summarize current trends in the identification and determination of bioactive compounds in mandarin.

Chitosan-based layer-by-layer edible coatings application for the preservation of mandarin fruit bioactive compounds and organic acids.

The layer-by-layer application of biopolymeric coatings to mandarin fruits as a postharvest treatment to improve fruit coating efficacy has been reported. A single 1 % (w/v) chitosan application was evaluated, and polyelectrolyte complexes such as 1.5 % (w/v) alginate/chitosan, 1 % (w/v) hydroxypropyl methylcellulose/chitosan, and 0.2 % (w/v) locust bean gum/chitosan were applied to mandarin fruits. The quality of coated mandarin fruits was observed at temperatures: 20 ± 2 °C (up to 10 days) and 5 °C (up to 28 days). Changes in the fruit metabolism were observed by evaluating bioactive compounds (polyphenolic compounds and flavonoids), antioxidant activity, and organic acids during the preservation of mandarin fruits. All of the tested combinations of layer-by-layer coatings significantly impacted the quality of mandarin fruits throughout storage, both at room temperature and cold storage, respectively. The overall best performance was observed for a layer-by-layer hydroxypropyl methylcellulose/chitosan coating in terms of visual aspects, bioactive compounds, antioxidant activity, and organic acids content.

Encapsulation of Rosemary Extracts using High Voltage Electrical Discharge in Calcium Alginate/Zein/Hydroxypropyl Methylcellulose Microparticles.

The increased demand for functional food with added health benefits is directing industrial procedures toward more sustainable production of naturally added bioactive compounds. The objective of this research was to investigate the potential of bioactive compounds from rosemary extract obtained using high-voltage electrical discharge as a green extraction method, for microencapsulation as a protective method for future application in functional food. Four types of microparticles were made via the ionic gelation method using alginate (Alg), zein (Z), and hydroxypropyl methylcellulose (HPMC) biopolymers and were analyzed considering the physicochemical properties. The diameter of dry microparticles ranged from 651.29 to 1087.37 µm. The shape and morphology analysis of microparticles showed that the obtained microparticles were quite spherical with a granular surface. The high encapsulation efficiency was obtained with a loading capacity of polyphenols up to 11.31 ± 1.47 mg GAE/g (Alg/Z microparticles). The microencapsulation method showed protective effects for rosemary polyphenols against pH changes during digestion. Specifically, the addition of both zein and HPMC to calcium-alginate resulted in microparticles with a prolonged release for better availability of polyphenols in the intestine. This research background indicates that the release of rosemary extract is highly dependent on the initial biopolymer composition with high potential for further functional food applications.

Novel Zinc/Silver Ions-Loaded Alginate/Chitosan Microparticles Antifungal Activity against Botrytis cinerea.

Addressing the growing need for environmentally friendly fungicides in agriculture, this study explored the potential of biopolymer microparticles loaded with metal ions as a novel approach to combat fungal pathogens. Novel alginate microspheres and chitosan/alginate microcapsules loaded with zinc or with zinc and silver ions were prepared and characterized (microparticle size, morphology, topography, encapsulation efficiency, loading capacity, and swelling behavior). Investigation of molecular interactions in microparticles using FTIR-ATR spectroscopy exhibited complex interactions between all constituents. Fitting to the simple Korsmeyer-Peppas empirical model revealed the rate-controlling mechanism of metal ions release from microparticles is Fickian diffusion. Lower values of the release constant k imply a slower release rate of Zn2+ or Ag+ ions from microcapsules compared to that of microspheres. The antimicrobial potential of the new formulations against the fungus Botrytis cinerea was evaluated. When subjected to tests against the fungus, microspheres exhibited superior antifungal activity especially those loaded with both zinc and silver ions, reducing fungal growth up to 98.9% and altering the hyphal structures. Due to the slower release of metal ions, the microcapsule formulations seem suitable for plant protection throughout the growing season. The results showed the potential of these novel microparticles as powerful fungicides in agriculture.

Increasing the lycopene content and bioactive potential of tomato fruits by application of encapsulated biological and chemical agents.

Plant secondary metabolites play an important part in the human diet. This research investigated the effect of alginate microspheres loaded with chemical (calcium or copper ions) or chemical and biological (Trichoderma viride) agents on plant secondary metabolites synthesis of two tomato varieties ('Vasanta' and 'Abellus') in two types of greenhouse cultivation, hydroponic and soil. Targeted and controlled release of active agents facilitates the root of plants to respond to the encapsulated agents and stimulate the synthesis of investigated plant metabolites both in hydroponic and soil types of cultivation. A significant increase in lycopene (up to 230%), total polyphenols content (up to 61%), and the overall antioxidant activity (up to 77%) of the tomato fruits was found for all of the treatments, respectively. Encapsulated chemical and biological agents remarkably stimulate the synthesis of plant secondary metabolites in tomato fruits indicating its great potential in the production of value-added foods.

Encapsulation of two fermentation agents, Lactobacillus sakei and calcium ions in microspheres.

Alginate microspheres loaded with two fermentation active agents, calcium cations and strain LS0296 identified as Lactobacillus sakei, have been prepared and characterized. The role of calcium cation is twofold, it acts as gelling cation and as fermentation active agent. Encapsulation and the presence of calcium ions in the same compartment do not inhibit the activity of LS0296. Molecular interactions in microspheres are complex, including mainly hydrogen bonds and electrostatic interactions. In vitro calcium cations and strain LS0296 release profiles were fitted to the Korsmeyer-Peppas empirical model. The calcium cation release process is driven at first by Fickian diffusion through microspheres and then by anomalous transport kinetics. The in vitro LS0296 release process is driven by Fickian diffusion through microspheres showing a much slower releasing rate than calcium cations. The release of LS0296 strain is followed by a decrease in the pH value. Results obtained give us a new insight into complex interactions between bacterial cultures and microsphere constituents. Prepared formulations of calcium alginate microspheres loaded with LS0296 could be used as a new promising tool and a model for different starter cultures encapsulation and use in the production of fermented foods.

N-Alkylated C-Glycosyl Amino Acid Derivatives: Synthesis by a One-Pot Four-Component Ugi Reaction.

C-glycosides represent an important group of naturally occurring glycosylation derivatives but are also efficient mimetics of native O-glycosides. Here, a one-pot four-component methodology is described toward a library of N-alkylated C-glycosyl amino acid derivatives comprising seven different isopropylidene-protected carbohydrate units. The applied methodology tolerates different amines and isocyanides and provides access to Ugi products in yields up to 85 %. X-ray analysis of selected products bearing three different carbohydrate motifs and comparison of their crystal structures with similar ones deposited in Cambridge Crystallographic Database revealed that four structures adopt different conformations, mostly not typical for peptide structures. This property opens the possibility to exploit here described N-alkylated C-glycosyl amino acid derivatives as templates to access different biotic and abiotic secondary structures.

The mechanism behind the selection of two different cleavage sites in NAG-NAM polymers.

Peptidoglycan is a giant molecule that forms the cell wall that surrounds bacterial cells. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected by ß-(1,4)-glycosidic bonds and cross-linked with short polypeptide chains. Owing to the increasing antibiotic resistance against drugs targeting peptidoglycan synthesis, studies of enzymes involved in the degradation of peptidoglycan, such as N-acetylglucos-aminidases, may expose new, valuable drug targets. The scientific challenge addressed here is how lysozymes, muramidases which are likely to be the most studied enzymes ever, and bacterial N-acetylglucosaminidases discriminate between two glycosidic bonds that are different in sequence yet chemically equivalent in the same NAG-NAM polymers. In spite of more than fifty years of structural studies of lysozyme, it is still not known how the enzyme selects the bond to be cleaved. Using macromolecular crystallography, chemical synthesis and molecular modelling, this study explains how these two groups of enzymes based on an equivalent structural core exhibit a difference in selectivity. The crystal structures of Staphylococcus aureusN-acetylglucosaminidase autolysin E (AtlE) alone and in complex with fragments of peptidoglycan revealed that N-acetylglucosaminidases and muramidases approach the substrate at alternate glycosidic bond positions from opposite sides. The recognition pocket for NAM residues in the active site of N-acetylglucosaminidases may make them a suitable drug target.