The Application of Protein Concentrate Obtained from Green Leaf Biomass in Structuring Nanofibers for Delivery of Vitamin B12.

Nanofibers made of natural proteins have caught the increasing attention of food scientists because of their edibility, renewability, and possibility for various applications. The objective of this study was to prepare nanofibers based on pumpkin leaf protein concentrate (LPC) as a by-product from some crops and gelatin as carriers for vitamin B12 using the electrospinning technique. The starting mixtures were analyzed in terms of viscosity, density, surface tension, and electrical conductivity. Scanning electron micrographs of the obtained nanofibers showed a slight increase in fiber average diameter with the addition of LPC and vitamin B12 (~81 nm to 109 nm). Fourier transform infrared spectroscopy verified the physical blending of gelatin and LPC without phase separation. Thermal analysis showed the fibers had good thermal stability up to 220 °C, highlighting their potential for food applications, regardless of the thermal processing. Additionally, the newly developed fibers have good storage stability, as detected by low water activity values ranging from 0.336 to 0.376. Finally, the release study illustrates the promising sustained release of vitamin B12 from gelatin-LPC nanofibers, mainly governed by the Fickian diffusion mechanism. The obtained results implied the potential of these nanofibers in the development of functional food products with improved nutritional profiles.

Broccoli, Amaranth, and Red Beet Microgreen Juices: The Influence of Cold-Pressing on the Phytochemical Composition and the Antioxidant and Sensory Properties.

The aim of this study was to analyze in detail the phytochemical composition of amaranth (AMJ), red beet (RBJ), and broccoli (BCJ) microgreens and cold-pressed juices and to evaluate the antioxidant and sensory properties of the juices. The results showed the presence of various phenolic compounds in all samples, namely betalains in amaranth and red beet microgreens, while glucosinolates were only detected in broccoli microgreens. Phenolic acids and derivatives dominated in amaranth and broccoli microgreens, while apigenin C-glycosides were most abundant in red beet microgreens. Cold-pressing of microgreens into juice significantly altered the profiles of bioactive compounds. Various isothiocyanates were detected in BCJ, while more phenolic acid aglycones and their derivatives with organic acids (quinic acid and malic acid) were identified in all juices. Microgreen juices exhibited good antioxidant properties, especially ABTS•+ scavenging activity and ferric reducing antioxidant power. Microgreen juices had mild acidity, low sugar content, and good sensory acceptability and quality with the typical flavors of the respective microgreen species. Cold-pressed microgreen juices from AMJ, RBJ, and BCJ represent a rich source of bioactive compounds and can be characterized as novel functional products.

Ultrasonication for production of nanoliposomes with encapsulated soy protein concentrate hydrolysate: Process optimization, vesicle characteristics and in vitro digestion.

This study presents the state-of-art research about the assembly of soy proteins in nanocarriers, liposomes, and its design includes different physicochemical strategies and approaches: two-step enzymatic hydrolysis of soy concentrate, hydrolysate encapsulation by using phospholipids and cholesterol, and application of ultrasonication. Achieved results revealed that ultrasonication, together with cholesterol addition into phospholipid layers, improved the stability of nanoliposomes, and a maximum EE value of 60.5 % was obtained. Average size of peptide-loaded nanoliposomes was found to be from 191.1 to 286.7 nm, with a ζ potential of -25.5 to -34.6 mV, and a polydispersity index of 0.250-0.390. Ultrasound-assisted encapsulation process did not lead to a decrease in the antioxidant activity of the trapped peptides. FTIR has indicated an effective hydrophobic interaction between phosphatidylcholine and hydrolysate peptides. TEM and SEM have confirmed the spherical nanocarrier structure and unilamelarity. Prolonged gastrointestinal release and stability of peptides have been enabled by liposome nanocarriers.

Immobilization of Horseradish Peroxidase on Magnetite-Alginate Beads to Enable Effective Strong Binding and Enzyme Recycling during Anthraquinone Dyes' Degradation.

The aim of this study was to investigate covalent immobilization of horseradish peroxidase (HRP) on magnetic nanoparticles (Mag) encapsulated in calcium alginate beads (MABs) for color degradation, combining easy and fast removal of biocatalyst from the reaction mixture due to its magnetic properties and strong binding due to surface alginate functional groups. MABs obtained by extrusion techniques were analyzed by optical microscopy, FEG-SEM and characterized regarding mechanical properties, magnetization and HRP binding. HRP with initial concentration of 10 mg/gcarrier was successfully covalently bonded on MABs (diameter ~1 mm, magnetite/alginate ratio 1:4), with protein loading of 8.9 mg/gcarrier, immobilization yield 96.9% and activity 32.8 U/g. Immobilized HRP on MABs (HRP-MABs) was then used to catalyze degradation of two anthraquinonic dyes, Acid Blue 225 (AB225) and Acid Violet 109 (AV109), as models for wastewater pollutants. HRP-MABs decolorized 77.3% and 76.1% of AV109 and AB225, respectively after 15 min under optimal conditions (0.097 mM H2O2, 200 mg of HRP-MABs (8.9 mg/gcarrier), 0.08 and 0.1 g/mg beads/dye ratio for AV109 and AB225, respectively). Biocatalyst was used for 7 repeated cycles retaining 75% and 51% of initial activity for AB225 and AV109, respectively, showing potential for use in large scale applications for colored wastewater treatment.

Design and characterization of whey protein nanocarriers for thyme essential oil encapsulation obtained by freeze-drying.

Innovative coating powders, based on whey protein concentrate (10-15 wt%) as native (WPC) or denatured protein (d-WPC), solely or in combination with alginate (0.75 wt%, AL), containing thyme essential oil, were produced using the freeze-drying technique. The impact of individual components (protein, alginate and oil) as well as the effect of heat-induced protein denaturation, was resolved regarding physicochemical, thermal and morphological properties of powders. High product yield (∼100%), particle size (223-257 nm), low moisture content (0.10-0.13%) and zeta potential (-19 to -25.6 mV) were determined for all samples. Strong antimicrobial activity of thyme oil nanocarriers against foodborne pathogens was demonstrated. Thermogravimetric analysis (TGA) indicated enhanced thermal stability of encapsulated oil. The most specific bands of structural compounds were identified in Raman spectra of the tested formulations, but principal component analysis (PCA) on recorded spectra was necessary to show the differences between carriers of different wall materials.

The Structuring of Sage (Salvia officinalis L.) Extract-Incorporating Edible Zein-Based Materials with Antioxidant and Antibacterial Functionality by Solvent Casting versus Electrospinning.

In this study, in order to develop zein-based, edible, functional food-contact materials in different forms incorporating sage extract (10, 20, and 30%), solvent casting and electrospinning were employed. The study aimed to assess the effects of the applied techniques and the extract's incorporation on the materials' properties. The solvent casting generated continuous and compact films, where the extract's incorporation provided more homogenous surfaces. The electrospinning resulted in non-woven mats composed of ribbon-like fibers in the range of 1.275-1.829 µm, while the extract's incorporation provided thinner and branched fibers. The results indicated the compatibility between the materials' constituents, and efficient and homogenous extract incorporation within the zein matrices, with more probable interactions occurring during the solvent casting. All of the formulations had a high dry matter content, whereas the mats and the formulations incorporating the extract had higher solubility and swelling in water. The films and mats presented similar DPPH• and ABTS•+ radical scavenging abilities, while the influence on Staphylococcus aureus and Salmonella enterica subsp. enterica serovar Typhimurium bacteria, and the growth inhibition, were complex. The antioxidant and antibacterial activity of the materials were more potent after the extract's incorporation. Overall, the results highlight the potential of the developed edible materials for use as food-contact materials with active/bioactive functionality.

Impact of Different Enzymatic Processes on Antioxidant, Nutritional and Functional Properties of Soy Protein Hydrolysates Incorporated into Novel Cookies.

Soy protein concentrate (SPC) was hydrolyzed using several commercial food-grade proteases (Alcalase, Neutrase, papain, Everlase, Umamizyme, Flavourzyme) and their combination to obtain promising ingredients in the manufacture of functional bakery products. In all cases, the hydrolysis caused nutritional, sensory, and rheological changes in SPC, as well as protein structural changes like increased surface hydrophobicity and content of exposed SH groups with the magnitude of these changes depending on enzyme specificity. The hydrolysis with the combination of Neutrase and Flavourzyme (NeuFlav) increased essential amino acid content by 9.8% and that of Lys by 32.6% compared to SPC. This hydrolysate showed also significant antioxidant activities including ABTS and superoxide anion scavenging activity and metal-chelating ability. The addition of all hydrolysates in wheat flour decreased water adsorption and increased development time to some extent due to gluten network weakening, but also decreased the rate of starch retrogradation, contributing to the increase of the shelf-life of bakery products. The NeuFlav tasted less bitter than other hydrolysates, while E-nose provided a discrimination index of 93 between control and hydrolysates. It appeared that the addition of the NeuFlav hydrolysate in a cookie formulation improved protein content and nutritional quality and directed to its higher general consumer acceptability than cookies formulated with only wheat flour.

Freeze vs. Spray Drying for Dry Wild Thyme (Thymus serpyllum L.) Extract Formulations: The Impact of Gelatin as a Coating Material.

Freeze drying was compared with spray drying regarding feasibility to process wild thyme drugs in order to obtain dry formulations at laboratory scale starting from liquid extracts produced by different extraction methods: maceration and heat-, ultrasound-, and microwave-assisted extractions. Higher total powder yield (based on the dry weight prior to extraction) was achieved by freeze than spray drying and lower loss of total polyphenol content (TPC) and total flavonoid content (TFC) due to the drying process. Gelatin as a coating agent (5% w/w) provided better TPC recovery by 70% in case of lyophilization and higher total powder yield in case of spray drying by diminishing material deposition on the wall of the drying chamber. The resulting gelatin-free and gelatin-containing powders carried polyphenols in amount ~190 and 53-75 mg gallic acid equivalents GAE/g of powder, respectively. Microwave-assisted extract formulation was distinguished from the others by a higher content of polyphenols, proteins and sugars, higher bulk density and lower solubility. The type of the drying process mainly affected the position of the gelatin-derived -OH and amide bands in FTIR spectra. Spray-dried formulations compared to freeze-dried expressed higher thermal stability as confirmed by differential scanning calorimetry analysis and a higher diffusion coefficient; the last feature can be associated with the lower specific surface area of irregularly shaped freeze-dried particles (151-223 µm) compared to small microspheres (~8 µm) in spray-dried powder.

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate.

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio-derived materials for delivery of dexamethasone-sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/-HESI-MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts' surface morphology by field emission-scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046-1055, 2016.

Erythrocyte membranes from slaughterhouse blood as potential drug vehicles: Isolation by gradual hypotonic hemolysis and biochemical and morphological characterization.

The present study was aimed at investigating the effect of isolation process-gradual hypotonic hemolysis on chosen parameters of the erythrocyte membranes (ghosts) originating from bovine and porcine slaughterhouse blood. The estimation of the gradual hypotonic hemolysis as a drug loading procedure for the erythrocyte ghosts was performed as well. Based on the results derived from analysis of the osmotic properties of the erythrocytes, the gradual hemolysis was performed with high volume of erythrocytes and 35mM hypotonic sodium-phosphate/NaCl, enabling >90% of hemolysis for both types of erythrocytes. Detailed insight into ghosts' morphology by field emission-scanning electron microscopy revealed a distortion from erythrocyte shape and an altered surface texture with increased bilayer curvature for both samples. Compared to erythrocytes, an average diameter of ghosts from both type of erythrocytes decreased for only about 10%. The reported unidispersity of the isolated ghosts is of great importance for their potential application as vehicles of active compounds. Gradual hemolysis did not lead to substantial loss of cholesterol and membrane/cytoskeleton proteins. This result indicated the ghosts' possibility to mimic the chemical and structural anisotropic environment of in vivo cell membranes, which is of significance for drug diffusion and partition coefficients. Induced shift of phosphatidylserine to external surface of the ghosts demonstrated their potential application as vehicles for targeted drug delivery to cells of reticuloendothelial system. Ultra high-performance liquid chromatography and Fourier transform infrared spectroscopy revealed the presence of a drug model - dexamethasone-sodium phosphate, and its interaction with structural components in both types of erythrocyte ghosts.

Epidemiology of end-stage renal disease and hemodialysis treatment in Serbia at the turn of the millennium.

The study presents the epidemiological features of patients treated with renal replacement therapy (RRT) in Serbia from 1997 to 2009 and compares the results of hemodialysis treatment in 1999 and 2009. Epidemiological data were obtained from the National Registry of RRT patients and data on hemodialysis treatment from special surveys conducted in 1999 and 2009. Within the period 1997-2009 the incidence of patients on RRT increased from 108 to 179 per million population (pmp), prevalence rose from 435 to 699 pmp, while mortality rate fell from 20.7% to 16.7%. The frequency of patients with glomerulonephritis decreased, while that of patients with diabetes and hypertensive nephropathy increased. In late 2009 there were 5208 patients receiving RRT in Serbia. Within the examined period new hemodialysis and reverse osmosis equipment were purchased, high-flux dialyzers with synthetic membranes were increasingly used and the number of patients receiving hemodiafiltration increased to 17.6%. Kt/V greater than 1.2 was recorded in 16% of the patients in 1999 but 52% in 2009. Options for correction of anemia and mineral disorders have also improved. The percentage of patients with HbsAg (13.8% vs. 4.8%) as well as anti-hepatitis C virus antibodies positive patients (23.2% vs. 12.7%) was significantly lower in 2009 than in 1999. Both the incidence and prevalence of RRT patients in Serbia are rising continuously, while the mortality rate is falling. More favorable conditions for dialysis treatment have brought about significant improvement in the results over the last 10 years.

[Restless legs syndrome prevalence in patients on chronic hemodialysis in central Serbia].

BACKGROUND/AIM: Restless legs syndrome (RLS) is a sensorymotor disorder and the core feature of this syndrome is a distressing, irresistible need or urge to move the legs. RLS is one of the most troublesome conditions experienced by endstage renal disease patients treated with regular hemodialysis, and is significantly associated with their increased mortality. The aim of this study was to assess the prevalence of RLS in the patients with end-stage renal failure in the three dialysis centers in central Serbia, and to investigate the association of the syndrome with clinical characteristics and biochemical variables. METHODS: A cross-sectional multicentric study in which 166 consecutive hemodialysis patients were evaluated using a standard questionnaire based on diagnostic criteria elaborated by the International Restless Legs Syndrome Study Group was performed. All patients were reinterviewed and clinically examined by a qualified neurologist. The data on the patients with and without RLS were compared. RESULTS: The prevalence of RLS was 22.96% (95% CI, 16.57% to 29.35%). There was no significant difference in duration of hemodialysis and clinical characteristics when we compared the patients with RLS with those without RLS. We found significantly higher levels of blood urea nitrogen and creatinine and lower level of Kt/V index in the patients with RLS, which might point to inadequate dialysis in these patients. CONCLUSION: In our study the prevalence of RLS in the patients on the regular hemodialysis was 22.96%. The obtained results are similar to those of other studies using the same methodology in the white population on the regular hemodialysis.