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The study deals with the evaluation of the onion peel potential, as a source of functional compounds (pectin and quercetin) for the formulation of polysaccharide-based delivery systems and innovative food products. In the first step, quercetin and its derivates, as representative lipophilic polyphenolic antioxidants, were extracted from onion peel by conventional (20 °C and 80 °C) and ultrasound assisted techniques using 70 % aqueous ethanol solution. Afterwards, the obtained quercetin- rich extracts were encapsulated in form of hydrogel particles by ionic gelation, using alginate and pectin as carriers. For that purpose pectin was previously extracted from onion peel using acid hydrolysis, followed by dialysis, evaporation and freeze drying. Pectin was combined with alginate (20:80, 30:70 and 40:60, v/v) for the formulation of quercetin- rich hydrogel particles. The physico- chemical and morphological properties, encapsulation efficiency of polyphenols and quercetin release profile from obtained particles in simulated gastric and intestinal fluids were examined. Although there were no marked differences between encapsulation efficiency (EETPC>93 %) in developed delivery systems, the highest content of quercetin and its derivatives was determined in alginate-pectin (80:20) particles. The obtained results confirmed a good application potential of quercetin and pectin, both derived from onion peel as compounds for encapsulation- enrichment of food products.

Cells of our organism are permanently exposed to different endogenous and exogenous oxidants, originated from basal metabolism, immune reactions, xenobiotics, detoxyfing processes and intermediates formed during biotransformations. At cellular level, oxidants cause DNA damage, oxidation of the proteins and destruction of lipids by lipid peroxidation chain reactions. On the other hand, numerous chemicals (of exogenous or endogenous origin) easily interacts with reactive oxidative species, preventing macromolecules destruction and consequently, damages of the cells and tissues. Significant role in this oxidative/antioxidative balance play bioactive compounds from plants, fruits or vegetables. Under certain circumstances, these compounds can decrease level of oxidative damage, but at the same time, under some other circumstances they can enhance prooxidative response. There are different assays for measurement of the antioxidant status and oxidative damage. The simplest ones are chemical in vitro reactions and tests in cell cultures. They can yield useful information about mechanisms of action, but extrapolation to effects of dietary antioxidants in vivo is not simple and often it may be incorrect, because uptake from the gastrointestinal tract, influence of microflora and metabolism are not considered. In this presentation some of in vitro models for detection of prooxidative/antioxidative effects will be presented, including the most common biomarkers that are measured in order to determine changes of cellular macromolecules that are the first step in the development of degenerative diseases.

Nowadays, there is a global interest in the technological use of agro-industrial residues as a renewable source of food or food supplements. In order to improve valorisation of sugar beet pulp as a pectin- rich secondary raw material, the aim of this study was to investigate the delivery potential of pectin extracted from sugar beet pulp (SBP) in the formulation of functional edible films. Beside pectin, new developed edible films, based on yellow tea as a source of bioactive compounds, were additionally enriched with fenugreek and rice proteins. In order to remove the hydrophilic compounds of low molecular weight, extraction of SBP pectin was performed using 70 % ethanolic solution, and afterwards pectin from alcohol insoluble residue was extracted by acid hydrolysis, followed by dialysis, evaporation and freeze drying. Physical properties of proteins and isolated SBP pectin, as well as physico-chemical, mechanical, bioactive and sensory properties of the formulated films were determined. Based on the obtained results, the highest content of total polyphenols (106.22 mg GAE/g film) and individual bioactive compounds (gallic acid, catechin, epigallocatechin and caffeine), as well as the highest value of antioxidant capacity (1.89 mmol Trolox/g film) were determined in the film enriched with rice proteins. Although yellow tea markedly improved the functional potential of all formulated films, pectin film without proteins exhibited the best mechanical and sensory properties, in terms of tensile strength, elasticity, acceptability, colour, transparency and appearance.

BRIEF INTRODUCTION: During recent years, innumerable scientific studies have been focused on examination of bioactive compounds with antioxidant properties, aimed for their use in prevention and treatment of different health issues. However, research and final application has mostly been dedicated to single compounds, without taking into account the potential synergistic effects of a multi-component mixture of different bioactive compounds. MATERIALS AND METHODS: In this study, antioxidant properties of single and the combination of chemically different groups of bioactive compounds ; polyphenol quercetin, ascorbic acid, aminoacids lysine and carnitine were assesed to evaluate whether synergistic interactions and effects occur. For that purpose, in vitro methods of antioxidant capacity determination by free radical scavenging assays (ABTS and DPPH assays) and by cytotoxicity and reactive oxygen species (ROS) formation on model human laryngeal carcinoma (HEp2) and colon cancer (CaCo2) cell lines were employed. Electron paramagnetic resonance (EPR) spectroscopy was introduced as an advanced technique to confirm the kinetics of a model spin-probe (TEMPO) scavenging by the evaluated antioxidant mixture. RESULTS: The use of ascorbic acid enabled to adjust the pH and enhance the solubility of quercetin in water, resulting with formulation of multi-component aqueous mixture. Synergistic effect in terms of enhancement of antioxidant capacity of formulated mixture was achieved (with regard to theoretical values). The formulated mixture exhibited cytoprotective and antioxidant activity on HEp2 cells, which was attributed to the same effect of quercetin and ascorbic acid, while single amino acids lysine and carnitine exhibited no significant effect on the survival of HEp2 and CaCo2 cells. EPR measurements revealed that the enhanced antioxidant effect of bioactive mixture does not differ significantly from plain ascorbic acid, indicating that the ROS scavenging of the formulated mixture would most likely be attributed to ascorbic acid and its prominent antioxidant properties. CONCLUSIONS: Due to the prevalent activity of ascorbic acid, further dose-response optimization is required to allow the formulation of functional, nutraceutical antioxidant mixture.

Proceedings contains articles presented at Conference divided into sections: open lecture (1), chemical analysis and synthesis (3), chemical and biochemical engineering (8), food technology and biotechnology (8), medical chemistry and pharmacy (3), environmental protection (11) and meeting of young chemists (2).

In recent years utilizing biopolymer interactions, or crosslinking-assembled spherical structures, enabled the encapsulation and targeted delivery of numerous active ingredients. The self-assembly of proteins such as zein or casein micelles has already been explored as a vehicle for delivery of hydrophobic bioactive compounds, while the use of alginate-protein or chitosan-protein complexes has been introduced very recently, mostly for encapsulation of pharmaceutical model drugs. In this study we focus on using alginate, chitosan and proteins as biopolymer building blocks for encapsulation of quercetin, a representative poorly water-soluble lipophilic polyphenolic antioxidant. The approach of improving quercetin solubility in a mixture of ascorbic acid was evaluated, and compared to plain compounds based on their antioxidant capacity. Electrostatic complexation between alginate and chitosan and reinforcement with whey proteins was conducted and mediated additionally by temperature (70°C) and ionic cross-linking. The encapsulation of both quercetin and ascorbic acid was achieved, enabling to entrap up to 66% of quercetin and 54% of ascorbic acid. Macro-sized spherical particles of alginate-chitosan and whey proteins-chitosan were produced ranging up to 2 mm in size, with the use of chitosan enabling the production of smaller particles. Whey protein-chitosan particles were less spherical, but provided the most retarded, prolonged release of quercetin from the particles in simulated gastric and intestinal fluids.

Agro-industrial waste is considered a valuable secondary raw material for various industrial processes and the separation of various functional constituents due to its high availability and an interesting chemical composition, while simultaneously reducing environmental burden and costs. Taking into consideration the rapid expansion of the market and its increasing demands for functional foods, agro-industrial wastes show a great potential for application as new, non-food-competing sources for carbohydrate recovery and the production of new functional delivery systems. Since the previous carbohydrate analysis enabled the characterization of sugar beet pulp and onion peel as pectin-rich secondary raw materials, the aim of this study was to establish the extraction procedure with maximum pectin yields for both raw materials and to investigate the carrier potential of extracted pectin in the formulation of rosemary (Rosmarinus officinalis L.) based edible films and hydrogel microparticles. The extraction of pectin from secondary raw materials started using 70% (v/v) ethanolic solution, in order to remove the hydrophilic compounds of low molecular weight, especially the non-structural carbohydrates. In order to obtain the maximum pectin yields, the extraction solvents (HCl, EDTA), the pH and the temperature of the reaction mixture were varied. Different concentrations of pectin (1%, 2%, 3% and 4%) from sugar beet pulp and onion peel were used in order to formulate the rosemary based hydrogel particles by ionic gelation and edible films by solvent casting method. A comparative study of the physico-chemical, bioactive and sensory properties of the produced films and the microparticles was undertaken. The highest yield of onion peel pectin (25.14%) was obtained using the chelating agent (EDTA), undergoing the extraction process at 90 °C and pH 7.0, while sugar beet pulp pectin (18.24%) was extracted using HCl at 90 °C and pH 7.0. The best results regarding the morphology of the microparticles were obtained using 2% onion peel pectin. These microparticles exhibited a high encapsulation efficiency of rosemary total polyphenols (88.34 %) and hydroxycinnamic acids (92.00 %), as well as a high efficiency of retaining the antioxidant capacity (91.46 %). Although rosemary extract markedly improved the bioactive composition of all developed functional films, the film formulated using 4% sugar beet pulp pectin exhibited the best mechanical and sensory properties.

Nowadays, cancer has become a leading cause of fatal diseases worldwide. Recently, the emphasis is dedicated on investigation of natural, alternative remedies in treatment of cancer cell lines, where plant extracts, due to their rich bioactive composition and high biological activity, may play big role. In this study, biological effect of aqueous ginger (Zingiber officinale) extract (GE) on the human laryngeal cancer cell line (HEp2) and lung carcinoma cells (A549) was evaluated. Cytotoxic, prooxidative effect and lipid peroxidation were examined in cancer cell lines exposed to different concentrations of GE for 2 hours. Cell survival was determined by Neutral red test, while 2’, 7’-dichlorofluorescein- diacetate (DCFH-DA) method was used for determination of free radicals. Lipid peroxidation was evaluated by measuring concentration of generated malondialdehydes. GE showed significant (p

BRIEF INTRODUCTION: In recent years new technological trends in food production have been directed to the biorefinery approach of agro-industrial waste materials, resulting in the production of multitude of valuable products, especially functional ingredients such as biopolymers and bioactive compounds. Compositional analysis of cocoa bean husk (Theobroma cacao L.) conducted in this study revealed this agro-industrial waste material as the predominant source of carbohydrates (64% on a dry matter basis), but also proteins (16% dmb) as the macrostructural components. Additionally, distinguished by a very high content of bioactive compounds, especially polyphenolic antioxidants and methylxanthine theobromine, the potential of utilising these components in the formulation of new added value products focused this study on the recovery of cocoa bean husk functional compounds and their application in the formulation of alginate-based hydrogel particulate delivery systems. MATERIALS AND METHODS: A sequential recovery procedure was developed for obtaining a bioactive extract and carbohydrate and protein isolates from the raw material. Alkaline extraction-isoelectric precipitation at two temperatures (20°C and 70°C) was employed for recovery of the macrostructural fraction, whoose functional properties (solubility, foaming properties, electrical conductivity, fat absorption (FAC) and water holding capacity (WHC), emulsification activity) were characterized. RESULTS: In general, isolates obtained at higher extraction temperature (70°C) were characterized by higher recovery yields (7.9%), lower WHC, higher FAC, higher foaming capacities and stabilities. The recovered macrostructural isolates were used in combination with alginate to formulate particulate delivery systems encapsulating previously obtained bioactive extract. The isolate-reinforced encapsulates were not improved markedly in their physico-chemical properties but augmented the polyphenols retention in the particulate delivery matrix. CONCLUSIONS: The peformed characterization measurements point to expressed binding properties of polyphenols and interactions that those bioactive compounds display with proteins and polysaccharides, which indicates on the potential of utilizing the recovered cocoa bean husk constituents for development of novel, functional delivery systems.

Since most of the studies involving helichrysum (Helichrysum italicum) are focused on the properties and composition of the essential oil produced from this Mediterranean plant, the aim of this study was to develop edible films and hydrogels based on helichrysum water extract. Alginate and pectin (combined with whey protein, soy protein, and hemp protein) served as carriers for the formulation of biodegradable films and hydrogel beads. Films with various biopolymers and incorporated microparticles were prepared using the solvent casting method, while hydrogel particles were produced using ionic gelation. The bioactive profile (the content of total polyphenols, TPC and hydroxycinnamic acids, HCAC) and antioxidant capacity (AC) of the developed films and microparticles (as well as their combinations) were characterized using spectrophotometric methods. The physical (colour, thickness, dry matter content) properties of films and particles, and the sensory characteristics of the developed films were also evaluated. The highest TPC (31.31 mg GAE/g sample) and HCAC (27.20 mg CAE/g sample), as well as the highest AC (0.15 mmol TE/g sample), were determined in pectin based films. The addition of various proteins decreased the content of the examined bioactive parameters of formulated films, while the addition of hydrogel microparticles increased their thickness and dry matter content. The obtained results indicate a great application potential of developed biodegradable matrices as functional films for active food packaging.

Walnut shell is a lignocellulosic by-product material mainly used for combustion or as an abrasive. Due to the high content of xylan-type hemicellulose, it might be regarded as a promising novel source of xylan-derived functional products from renewable sources, such as xylooligosaccharides. The aim of this study was to determine the impact of delignification and varying alkali concentration on the extractability of xylan from walnut shells. Hemicellulose recovery was carried out by combining alkaline extraction (2M and 4M KOH + 20 mM NaBH4, for 4 h) with or without sodium chlorite-acetic acid delignification pretreatment (2 h or 4 h). The obtained fractions consisted of soluble and insoluble hemicellulose and insoluble cellulosic residue. The respective yields were determined gravimetrically, while their monomeric composition was determined by HPLC- PDA with pre-column derivatization using 3- methyl-1-phenyl-2-pyrazoline-5-one (PMP). Delignification enabled a significant increase in total hemicellulose yield (2.5 - 2.8-fold, compared to non-delignified samples), with a maximum obtained yield of 28.8 % of the original sample dry weight. Delignification time, as well as alkali concentration, only slightly influenced the yield. The highest total xylose recovery, 95 % of the initial content, was obtained for the 4 h delignified sample extracted for 4h with 4M KOH. It predominantly accounted for the extracted xylan (91.8 % of initially present xylose), of which 43 % was in insoluble form.

Bioactive features of diverse nutraceutical compounds as novel therapeutic supplements led to an increased interest of pharmaceutical and food domains in obtaining encapsulated dosage forms of those compounds that enable their wide applications. Polyphenolic antioxidants impart the largest number of evaluated and established antioxidant properties, however the possibility of synergistic effect of polyphenols with other bioactive compounds, such as amino acids and vitamins has not been evaluated properly. The present study was focused on defining the antioxidant potential of quercetin as a model polyphenolic compound in combination with ascorbic acid and amino acids L-lysine and taurine, aiming to obtain the most potent nutraceutical mixture that was encapsulated in alginate-based hydrogel particles by ionotropic gelation. The assessment of synergistic antioxidant properties by the ABTS, DPPH and FRAP assays revealed that although L-lysine and taurine water solutions did not exert antioxidant capacity, they enhanced the antioxidant potential of their binary mixtures with quercetin, providing up to 30% higher antioxidant capacity than just plain quercetin solutions. The combination of quercetin, L-lysine and ascorbic acid (each 150 mg/L), exhibited the most potent antioxidant properties, with up to 2-fold higher antioxidant capacity than just plain quercetin (150 mg/L) or 3-fold higher than plain ascorbic acid (150 mg/L). By encapsulating the stated synergistic mixture in 2% sodium alginate via calcium-induced crosslinking, 78% of rosmarinic acid was entrapped inside the freeze dried particles, along with 62% of vitamin C and 86% of L-lysine (established by spectrophotometric assays). Although L-lysine contributed less by its antioxidant properties, its cationic nature may have contributed to the augmented encapsulation efficiency of the active compounds by enhanced crosslinking due to the polyelectrolyte character of alginate-L-lysine matrix. The obtained results provide a novel approach to establishing innovative formulations of potent, functional nutraceutical dosage forms.

Nowadays, cancer has become a leading cause of fatal diseases worldwide. Recently, the emphasis is dedicated on investigation of natural, alternative remedies in treatment of cancer cell lines, where plant extracts, due to their rich bioactive composition and high biological activity may play big role. In this study, biological effect of aqueous ginger (Zingiber officinale) extract (GE) on the human larynx cancer cell line (Hep2) and lung carcinoma cells (A549) was evaluated. Cytotoxic, prooxidative effect and lipid peroxidation were examined in cancer cell lines exposed to different concentrations of GE for 2 hours. Cell survival was determined by Neutral red test, while 2’, 7’ dichlorofluoresceine diacetate (DCFDA) method was used for determination of free radicals. Lipid peroxidation was evaluated by measuring concentration of generated malondialdehides. GE showed significant decrease in cell survival for Hep2 and A549 in the highest concentration of 0.25 g/ml, while lower concentrations (0.05 and 0.125 g/ml) did not show cytotoxic effect. GE did not exhibit prooxidative effect, contrary, antioxidative effect of all concentrations on both cell lines was observed, where by increasing the concentration, free radical induction was decreased. Moreover, GE did not reveal effect on cell lipids. Obtained results confirmed markable health benefits of GE and justified its wide application in various pharmaceutical and food products.

Owing to the increased tendency of producing bioactives-enriched films and coatings for food applications, in this study the potential of formulating edible films from polymeric hydrocolloid materials (alginate, pectin, chitosan) in cocoa (Theobroma cacao L.) husk extract as the source of macroconstituent (polysaccharides) and bioactive compounds (polyphenols and methylxanthines) was investigated. Since cocoa husk extract represents the main cocoa processing by-product in the chocolate industry, valorization of this material by expoiting it as a source of added-value functional ingredients was examined. A comparative study of the physical, mechanical, bioactive and sensory properties of produced films, prior and after drying of films at 60°C, was undertaken, and the relation between specific properties (colour, sensory and mechanical properties) was deduced by correlation analysis. The effect of enrichment with polyphenolic compounds and theobromine derived from cocoa husk extract was determined by comparing the produced films with control films obtained only in distilled water. According to the obtained results, edible films were produced with thickness ranging from 30-100 μm, which was primarily in dependance of the solubilization media (water or cocoa husk extract), while the water and dry matter content were more affected by the employed biopolymer material. Control alginate film generally exhibited the best mechanical (highest tensile strenght - 6, 17 N and lowest deformation - 0, 73 %) and sensory properties, while the enriched chitosan film prepared in cocoa husk extract exhibited the poorest respective properties and the most potent thickness (104, 67 μm). By preparing the films in cocoa husk extract a significant increase of bioactive compounds content was achieved (even up to 3-fold higher) in relation to control films, while the bioactive properties remained preserved even after drying of films at 60°C. Enrichment of films in cocoa husk extract enabled to markedly enhance the bioactive content, but it negatively affected the physical, mechanical and sensory properties of produced films, which were better for control films (especially alginate one).

Recently, due to the low bioavailability and high instability of polyphenols, scientific community, as well as industry put much effort to find adequate solutions for polyphenolics protection, and revealed encapsulation technique as the most effective. Since bioactive profile of dandelion (Taraxacum officinale L.) is still insufficiently investigated, and data about its encapsulation are limited, the aim of this study was to characterize and immobilize dandelion leaves polyphenols in different alginate-based delivery systems using ionic gelation. In order to enhance the properties of alginate hydrogels, alginate (A) was combined with whey protein isolates (WPI), and green coffee (GC), which as material for delivery of bioactives has not been used before. Also, the influence of alginate concentration (4% and 5%) on the encapsulation efficiency of dandelion polyphenols was examined. Polyphenolic profile of dandelion leaves was determined using spectrophotometric and HPLC analyses. Obtained hydrogel particles were scanned for physico-chemical and morphological properties. Encapsulation efficiency was evaluated in terms of total polyphenols (TP) and hydroxycinnamic acids (HCA), while release kinetics of polyphenols was observed in simulated gastrointestinal conditions. HPLC analysis showed chicoric acid as dominant hydroxycinnamic acid in dandelion leaves (6.14 mg/g), followed by caftaric, chlorogenic and caffeic acids. Beads produced with lower concentration of alginate (4%) were characterized with smaller size, less hardness and lower elasticity. In general, the addition of additional coating to alginate increased particle size, where hydrogel beads produced with GC were the largest (2.66 and 2.80 mm). Morphology analysis revealed more spherical shape of hydrogels when higher concentration of alginate (5%) was used, where plain alginate beads were scanned as ones with the most regular shape. Encapsulation efficiency (EE) of TP and HCA was markedly enhanced after the reinforcement of alginate with WPI and GC. HCA of dandelion leaves were immobilized in a high content, where system A-WPI had the highest EE (98.99%), followed by A-GC (79.09%), when lower alginate concentration was used. The slowest release of HCA from formulated beads showed system enriched with GC (5%A), with continuously release during 140 min, up to 2.9 mg caffeic acid/g of bead. Implementation of green coffee to alginate, besides improving bioactive profile of obtained beads, showed very high potential for entrapment of dandelion polyphenols. These results encourage introduction of new, unused materials into delivery systems for encapsulation purposes.

In recent years utilizing biopolymer interactions, or crosslinking-assembled spherical structures, enabled the encapsulation and targeted delivery of numerous active ingredients. The self-assembly of proteins such as zein or casein micelles has already been explored as a vehicle for delivery of hydrophobic bioactive compounds, while the use of alginate-protein or chitosan-protein complexes has been introduced very recently, mostly for encapsulation of pharmaceutical model drugs. In this study we focus on using alginate, chitosan and proteins as biopolymer building blocks for encapsulation of quercetin, a representative poorly water-soluble lipophilic polyphenolic antioxidant. The approach of improving quercetin solubility in a mixture of ascorbic acid was evaluated, and compared to plain compounds based on their antioxidant capacity. Electrostatic complexation between alginate and chitosan and reinforcement with whey proteins was conducted and mediated additionally by temperature (T=70°C) and ionic cross-linking (Ca2+). The encapsulation of both quercetin and ascorbic acid was achieved, enabling to entrap up to 71% of quercetin and 55% of ascorbic acid. Micro-sized spherical particles of alginate-chitosan and whey proteins-chitosan were produced ranging up to 940 µm, with alginate-chitosan particles producing smaller particles. Whey protein-chitosan particles were less spherical, but provided much slower release of quercetin from the particles in simulated gastric and intestinal fluids.

Due to rich phytochemical composition of medicinal mushroom Ganoderma lucidum, especially ß-glucan-based polysaccharides and triterpenes, but as well polyphenols, amino acids, proteins, it is often used in various nutraceutical and functional food products. These products are lately formulated with microencapsulated forms of active compounds in order to prevent their degradation after oral consumption and during processing conditions. Since there is a lack in scientific data of Ganoderma polyphenolics, and especially their encapsulation, the aim of this study was to characterize and encapsulate polyphenols from aqueous extract of Ganoderma. In that order, ionic gelation of alginate (A), and its combination with whey protein isolates (WPI) and zein was used. Obtained hydrogel beads were scanned for physico-chemical and morphological properties, encapsulation efficiency of polyphenols and their release kinetics in simulated gastric and intestinal fluids. Total polyphenols (TP) exhibited 1.45 mg gallic acid equivalents/g of sample, among which total flavonoids amounted up to 45% of TP. The addition of WPI to alginate resulted with the reduction of particle size and spherical shape of beads, while beads formulated with zein were characterized as larger, with irregular morphology. Encapsulation efficiency of TP was determined in following order: 76.91% (A-WPI)

Rosemary (Rosmarinus officinalis L.), culinary and medicinal plant, due to its rich bioactive profile, especially polyphenolic antioxidants, has recently become of great interest as functional food ingredient. However, polyphenols reveal remarkable instability in the gastrointestinal tract after oral consumption, likewise during food processing and production. Therefore, the aim of this study was to examine the encapsulation potential of polyphenols from rosemary extract in alginate-based delivery systems using ionic gelation. In order to formulate delivery systems with enhanced encapsulation efficiency and slower release profile of polyphenolics, alginate (A, 2%, w/v) was combined with whey protein isolates (WPI), and natural materials which still have not been used for encapsulation purposes, cocoa powder (CP) and carob (C), respectively. Obtained hydrogel beads were evaluated for physico-chemical and morphological properties. Encapsulation efficiency (EE) was determined in terms of total polyphenols (TP) and hydroxycinnamic acids (HCA). The release kinetics of TP and HCA from beads was accompanied in simulated gastric (SGF) and intestinal (SIF) fluids. Among alginate beads encapsulating rosemary polyphenols, A-WPI beads resulted with the smallest particle size (1.7 mm), while the addition of CP and C provided larger particles (2.2 and 2.1 mm). In comparison to plain alginate beads, which were characterized with regular, spherical shape, the addition of other materials provided irregular beads and appearance. HCA, as the most representative polyphenolic group, were encapsulated in a high content, from 52.32% (A) to 91.16% (A-CP), indicating that introduction of an additional carrier to alginate improved its encapsulation properties. The same pattern was observed for release kinetics of TP, where A-CP and A-C had the most extended release of TP in SGF, followed by gradually release in SIF up to 140 min. Formulated carriers proved to be effective materials for immobilization of rosemary polyphenols, with potential of usage in various functional food products.

Medicinal plants like menthe and rosemary are traditionally used for disease treatment because of the chemical composition and antioxidant capacity. Different methods of extraction will result in different content of bioactive compounds in obtained extracts. Consequently, chemical and biological effect of extracted compounds will differ from one sample to another. Menthe extract has the highest amounts of polyphenols and the highest antioxidant capacity. Menthe extract showed cell type dependend antioxidative/prooxidative nature, while rosmary extract induced free radicals at higher concentrations.

Agro-industrial wastes comprise different types of plant residues that are generated as by-products on the field or in the factory. Such biomass is characterised by high carbohydrate content and nowadays is becoming an increasingly studied raw material for their isolation and re-utilisation within the sustainable production concepts in food technology and biotechnology. Due to the complexity of plant cell wall matrix, recovery of polysaccharides is a demanding task, whether the aim is obtaining satisfactory yields, preserving native structures or, often, both. The present study aimed to define a suitable procedure for sequential extraction of polysaccharides from sugar beet pulp (SBP), walnut shell (WS), cocoa bean husk (CBH), and pea pods (PP), as well as to establish a reliable method for compositional evaluation of the obtained fractions. Alcohol insoluble residue (AIR) was prepared by repeated extractions with 70% ethanol. AIR was subsequently subjected to hot diluted acid (HCl) extraction, delignification with NaClO2 and CH3COOH and finally alkaline extraction with NaOH. Yields of the obtained fractions were measured gravimetrically, while the composition using developed HPLC-UV technique with 3-methyl-1-phenyl-5-pyrazoline-2-one pre-column labeling, after complete hydrolysis. Three fractions were obtained, namely, pectic (P), hemicellulosic (H) and cellulosic (C), with fraction mass ratios P:H:C dependent on the sample and the employed extraction protocol. SBP exhibited the highest yield of pectins (18.24% dwb), while WS and PP of hemicelluloses (28.65% and 22.32% dwb). Monomeric composition evaluation revealed typical galacturonic acid dominance for pectic fractions of SBP and CBH, while their hemicelluloses were of very heterogeneous composition. The obtained hemicelluloses of WS and PP were xylan-type, with relative xylose content of nearly 90% and 80%, respectively. The established procedure is regarded as suitable for pectin and hemicellulose extraction due to high total xylan (71.53% for WS, 71.43% for PP) and galacturonan recoveries (88.84% for SBP) from the starting material.

Medicinal herbs and spices have traditionally been used as flavourings and they are widely dispersed as remedies in natural medicine, since they possess numerous beneficial properties on human health. Their positive sides in treatments of diseases are attributed to their rich phytochemical composition, with antioxidant polyphenols playing a key role. Bioactive characterization and biological effect of aqueous mint (Mentha piperita L.) extract on human larynx cancer cell line (Hep2) and lung carcinoma cells (A549) were evaluated in this study. Dried mint leaves were prepared by two conventional techniques, simulating infusion (80°C) and decoction (100°C) preparation techniques. Mint extracts (MEs) were characterized for their proximate composition, contents of polyphenolic compounds and soluble polysaccharides and antioxidant capacity (AC). Polyphenolics determination included spectrophotometric analyses of total polyphenols and flavonoids, flavan-3-ols, hidroxycinnamic acids, flavons, proanthocyanidins, tannins, while for determination of specific polyphenolic high-performance liquid chromatography (HPLC) analysis was applied. AC was estimated spectrophotometrically by ABTS (2, 2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), DPPH (2, 2-diphenyl-1-picrylhydrazyl) and FRAP (Fe(III)-3-pyridyl-triazine) assays. The ME characterized by higher polyphenolic yield was freeze dried and estimated for cytotoxic and prooxidative activities on larynx and lung carcinoma cells lines after two hours of treatment. Cell survival percentage was determined by Neutral red (NR) test, and free radicals level was measured by 2", 5" dichlorofluoresceine diacetate (DCFDA) method. ME exhibited high total polyphenol content, ranging from 60.64-70.04 mg gallic acid equivalents/g of dry sample weight. ME prepared by lower extraction temperature was revealed as more efficient in terms of providing a higher polyphenolic yield and AC, especially with regard to tannins content, which significantly could influence the sensory properties of herbal infusions. HPLC assay revealed rosmarinic acid as the most aboundant phenolic acid. Cytotoxicity results showed that bioactive compounds decreased cell survival for 25% (Hep2), and for 75% (A549), pointing lung carcinoma cells as more susceptible to cytotoxic effect of ME. Free radicals were not induced in these cell lines, but on the contrary, lower concentrations decreased basal level of free radicals in comparison to negative control. The obtained results suggest that moderate intake of prepared mint extract, due to its bioactive composition and biological activity could be useful in different pharmaceutical or food applications.

The trend of using traditional knowledge and raw materials in food production has been recognised in recent years as a part of the transition from quantity to quality. Autochtonous plant species, often characterized with a highly valuable nutritive and bioactive composition, might be used for the purpose of adding value to food products, thereby improving their functionality and profitability. The consumption of black (Morus nigra L.) and red (Morus rubra L.) mulberry fruits dates long ago, but they are nowadays poorly utilized due to the extensively managed industrial food resources. The potential of using these raw materials as a starting point for their use in food production as dried ingredients, natural sweetners and even colourants was examined in the present study through the evaluation of their sugars, polyphenolic and anthocyanins content, antioxidative and sensory properties. For the purpose of compositional characterization, high performance liquid chromatography coupled with refractive index detection (HPLC-RID) was used to evaluate the contents of sugars, while the phytochemical constituents were assayed spectrophotometrically. To evaluate the potential of using these ingredients in confectionery applications, the fruits were dried by convective (60°C) and freeze drying, and their sensory properties were evaluated by employing quantitative descriptive analysis procedure. Up to 4.7 mg of gallic acid equivalents/g dry matter of total polyphenols in mulberry juice was determined, while flavonoids constitued up to 38% of that content. The content of anthocyanins expressed as cyanidin-3-glucoside equivalents amounted up to 0.06 mg/g dry matter, with a much higher content determined in black mulberry. The ranking of antioxidant properties corresponded to the ranking of total polyphenolic compounds and anthocyanins and exhibited a decreasing tendency from fresh to heat dried fruits. Both black and red mulberry fruits exhibited a favourable sugar composition, since sucrose was not detected, while fructose was the predominant sugar with up to 120 mg/g dry matter determined in black mulberry. Freeze dried black mulberry was generally preferred in terms of the sensory properties, especially taste and texture, with a higher preference of panelists for dried rather than fresh products. The obtained results imply that dried mulberry fruits may be used alone or as functional alternatives in production of other food, especially confectionery products, which is supported by their favourable sugar composition and high polyphenolic antioxidants content.

In this study, alginate-based carrier systems consisting of carageenan, pectin or chitosan were formulated in order to evaluate the potential of caffeine encapsulation (as the model drug), with the aim of achieving its prolonged release and better encapsulation efficiency. For that purpose, plain alginate, alginate-pectin and alginate-carageenan beads were formulated and the latter two additionally coated with chitosan or pectin to evaluate whether coating can improve the encapsulation attributes of obtained beads. Particle size, encapsulation efficiency, release profiles in water, simulated gastric (SGF) and intestinal fluids (SIF) and FTIR spectra of formulated hydrogel beads were determined. According to the obtained results alginate-carrageen as the immobilization system for caffeine provided the largest particles (2.32 mm), while employing chitosan or pectin coating around the formed particles provided larger and irregularly shaped beads. The highest encapsulation efficiency of caffeine was achieved using the alginate-pectin formulation (79.7%), while the introduction of chitosan coating slightly enhanced the ability to entrap this compound. The release study in water revealed similar caffeine release kinetics for all evaluated carrier systems, with the majority of caffeine being released from the beads already during the initial 5-10 minutes of experiment. More pronounced release patterns of caffeine were determined in beads placed in SGF and SIF in comparison to water as the release medium. The release of caffeine was much slower from the coated particles, especially for the pectin coated alginate-carageenan beads in SGF and chitosan coated alginate-pectin beads in SIF, where the release of caffeine occurred gradually during 2h, probably as the result of coating formation that retarded the diffusion of caffeine from the particles. The obtained results indicate that the employed blended carrier systems are efficient to achieve a high encapsulation efficiency of caffeine, but further optimization is needed to prolong the release of caffeine from the beads in a liquid medium.

Medicinal plants are the richest bio-resource of drugs of traditional medicine systems, modern medicines, nutraceuticals and food supplements, since they comprise numerous bioactive compounds, which can serve as new sources of ingredients for inclusion in functional food products. Herbally enhanced functional foods are already present on the market ; however, little scientific data exists regarding the development and efficacy of functional confectionery products enriched with herbal phytochemicals. The aim of this study was to evaluate the potential of using bioactive compounds from two traditional medicinal plants ; nettle (Urtica dioica L.) and olive leaf (Olea europaea L.) for improving the functional properties, especially the polyphenolic profile and antioxidant capacity of chocolates. Physical and sensory properties, bioactive content and antioxidant capacity of three types of chocolates (containing 30%, 38% and 72% cocoa parts), enriched with microencapsulated polyphenolic compounds from nettle and olive leaf extracts were investigated (by employing electrostatic extrusion as the immobilization technique). The application of hydrogel microencapsulated herbal extracts impaired the physical properties of enriched chocolates, as well as their appearance and texture attributes, while the addition of freeze-dried microcapsules significantly improved that characteristics. An inverse relation between the content of added hydrogel microcapsules and physical and sensory properties of enriched chocolates was established. The addition of 3% of hydrogel microcapsules resulted with harder chocolates with higher viscosity, rough and coarse product surface, providing sensory unacceptable products. By increasing the content of microencapsulated herbal extracts, polyphenolic compounds and antioxidant capacity increased, while no significant difference between the content of polyphenolic compounds and antioxidant capacity of enriched chocolates, depending on the state of microcapsules (hydrogel or freeze-dried) was determined. The application of microencapsulated herbal extracts enabled the production of chocolates with improved functional quality that ensured controlled delivery and stability of bioactive compounds in the end products during their predicted shelf life.

The aim of this study was to evaluate and compare the bioactive and nutritional profile of red raspberry (Rubus idaeus L.) affected by two non-thermal drying techniques, freeze drying and CO2 drying. In that order, optimization of CO2 drying conditions with the aim of achieving the highest quality of the final product was conducted. UV/Vis spectrophotometric methods were employed to determine the content of total polyphenols and flavonoids, as well as antioxidant capacity evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radical scavenging assays. Since anthocyanins are among the most important quality indicators of red raspberries, and their contribution to the appearance and visual acceptability is crucial, individual anthocyanins were identified and quantified by means of reversed phase high performance liquid chromatography, which was also applied to determine the ascorbic acid and sugar contents of differently dried red raspberries (HPLC-PDA, -RID). By comparing the drying efficiency of freeze drying and CO2 drying, both techniques resulted with deterioration of bioactive compounds and antioxidant capacity, where freeze drying provided better preservation efficiency, while degradation tendency in CO2 drying depended on processing conditions. Comparison of freeze drying and the best CO2 drying condition showed that freeze drying provided only slightly better preservation of bioactive compounds and better preservation of the antioxidant capacity. Five individual anthocyanins were determined, among which cyanidins where more abundant (cyanidin-3- sophoroside, cyanidin-3-rutinoside, cyanidin-3-glucoside), while pelargonidins (pelargonidin-3-rutinoside, pelargonidin-3-glucoside) were present in a much lower content, in both fresh and dried red raspberry samples. Among sugars, fructose, glucose, and sucrose were identified in all raspberry samples. According to the obtained results, CO2 drying with the most suitable processing parameters may be regarded as a good alternative to freeze drying.

As a consequence of the modern lifestyle often accompanied by inadequate diet, a need for additional daily supplementation in bioactive compounds is increasing. Traditional medicinal plants from Lamiaceae family, due to their rich bioactive composition and high antioxidant capacity can be used to enhance and improve the bioactive and sensory properties of food. Since goat milk is less preferred by consumers due to its specific sensory characteristics that are often regarded as undesirable, in this study the addition of 10 % of mint and rosemary extracts to goat milk was evaluated, with the aim of enhancing the bioactive composition and improving its sensory properties. For the characterization of bioactive profile of mint and rosemary extracts, and enriched goat milk, UV/Vis spectrophotometric (total polyphenols and flavonoids, total tannins and hidroxycinnamic acids) and high-performance liquid chromatography (HPLC-PDA) methods were applied. Antioxidant capacity was determined by ABTS (2, 2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and DPPH (2, 2-diphenyl-1-picrylhydrazyl) methods. Sensory properties of herbal extracts, as well as enriched goat milk were evaluated using quantitative descriptive analysis procedure. Mint extract was characterized with higher content of total polyphenols and flavonoids (4745.45 and 2581.82 mg gallic acid equivalents/L), hidroxycinnamic acids (835.77 mg caffeic acid equivalents/L), as well as antioxidant capacity (22.30 and 28.25 mM Trolox/L by ABTS and DPPH), while rosemary extract provided a higher content of total tannins (0.88 %). The most abundant and representative polyphenolic compound in both extracts was rosmarinic acid. Addition of both herbal extracts to goat milk resulted with a higher content of bioactive compounds and antioxidant capacity in comparison to plain goat milk. Although rosemary extract was preferred over mint according to the sensory panel, goat milk enriched with rosemary extract was scored lower when compared to mint-containing milk. The results confirm that mint and rosemary extracts are a rich source of bioactive antioxidants, indicating their potential for use in food production. The addition of both extracts to goat milk enhanced its bioactive composition and improved sensory properties.

Biologically active compounds although being native constituents of food, are not consumed in a sufficient extent as a consequence of irregular and unbalanced diet. Therefore increasing interest arises in exploiting naturally derived plant products as sources of bioactive ingredients, in order to increase their intake and positive health effects. Among plant materials, carob (Ceratonia siliqua L.) and cocoa husk (Theobroma cocoa L.) have in common a high content of dietary fibres and polysaccharides, but a lack of information exists regarding their antioxidant capacity and polyphenolic profile. In this study the bioactive composition and antioxidant properties, as well as the content of dietary fibers and sugars in carob and cocoa husk, affected by different extraction techniques (hydrolysis, ultrasonic, conventional) and solvents (aqueous mixtures of acetone and ethanol) was evaluated using spectrophotometric and high performance liquid chromatography (HPLC) methods. Cocoa husk exhibited higher total dietary fibre content, but lower content of fructose and glucose, in comparison to carob, which was also characterized with a significant content of sucrose. Both acid and alkali hydrolysis of raw materials provided the highest content of polyphenolic compounds and antioxidant capacity, which could be attributed to interferences in the measurement procedure and non-selectivity of employed reagents. By comparing non-hydrolysed samples, the highest total phenol content (TPC) was determined in extracts prepared using 80% acetone (carob-15.2 mg gallic acid equivalents/g DM, cocoa husk-4.8 mg GAE/g DM). The ranking of samples according to their total flavonoid content and antioxidant capacity corresponds to their TPC order (80% acetone > 50% ethanol > 70% ethanol). HPLC analysis revealed the prevalence of methylxanthines in cocoa husk and gallic acid and its derivatives in carob. The evaluated plant materials represent rich sources of carbohydrates, as well as polyphenolic antioxidants, while the content of these compounds is dependent on the employed extraction technique and solvent.

Health benefits of Goji berries (Lycium barbarum L.) consumption, recognized for centuries in traditional Chinese medicine, are increasingly becoming popular among the modern consumers, with bioactive polysaccharides and pigments being the most studied compounds so far. Due to the lack of data on other bioactive constituents, polyphenolic antioxidants in particular, dried nature of Goji fruit as the most popular marketing form as well as thermolability of polyphenolics, the effect of different drying techniques on the latter was studied. Upon selecting the most suitable extraction procedure (80% acetone assisted conventional extraction), HPLC analysis have shown vanillic acid derivatives as the most abundant components of both fresh and dried Goji berry extracts. Freeze drying enabled the best preservation of these compounds, while oven drying at 60°C was shown to be the most detrimental.

There has been an increasing interest in development and design of functional food products with an enhanced bioactive profile, deriving from various plant materials, especially medicinal plants. Yarrow (Achillea millefolium L.) is one of the most abundantly occuring species, widely used in folk medicine but also in modern medicine practice. Yarrow is stated to possess diaphoretic, antipyretic, hypotensive, astringent, diuretic and urinary antiseptic properties. Traditionally, it has been used for bruises, swellings, strains, fevers, common cold, essential hypertension, amenorrhoea, dysentery, diarrhoea, and specifically for thrombotic conditions with hypertension, including cerebral and coronary thromboses. The aim of this study was to evaluate the potential of using different forms (microencapsulated, freeze-dried and concentrated) of yarrow (Achillea millefolium L.) extract, for enhancing the polyphenolic profile and antioxidant capacity of chocolates. Physical and sensory properties, bioactive content and antioxidant capacity of three types of chocolates (containing 30%, 38% and 72% cocoa parts), enriched with 2% of microencapsulated (by employing electrostatic extrusion as the imobilization technique), freeze-dried and concentrated polyphenolic compounds from yarrow extract were investigated. The application of concentrated and hydrogel microencapsulated yarrow extracts impaired the physical properties of enriched chocolates, as well as their appearance and texture attributes, while the addition of freeze-dried extract significantly improved that characteristics. An inverse relation between the content of added concentrated extract and hydrogel microcapsules and physical and sensory properties of enriched chocolates was established. The addition of 2% of concentrated extract and hydrogel microcapsules resulted with harder chocolates and higher viscosity, and consequently sensorily unacceptable product. However, by increasing the content of concentrated yarrow extract, polyphenolic compounds and antioxidant capacity increased, while no significant difference between the content of polyphenolic compounds and antioxidant capacity of enriched chocolates, depending on the addition of microencapsulated or freeze-dried extracts was determined. The application of microencapsulated herbal extracts enables the production of chocolates with improved functional quality, that ensures controlled delivery and stability of bioactive compounds in the end product during their predicted shelf life.

Worldwide, basil (Ocimum basilicum L.) is one of the most important culinary herbal crops, and is produced for fresh or dry leaf, essential oil and seed markets. Apart from culinary value, basil has attracted considerable interest among researchers during the last decade due to its bioactive properties. Strong antioxidant activity, ascribed to high levels of polyphenolic compounds, as well as health beneficial properties, including anticancerogenic, antimicrobial, anticonvulsant and stimulative to digestion activities, support the importance of maintaining high levels of bioactive compounds of basil. In basil processing, most conventional drying techniques, such as air drying, use relatively high temperatures for substantial amount of time, causing losses of volatiles and flavour, changes in colour and texture and deterioration of bioactive substances, which results in lower quality of the final product. In that order, alternative non-thermal drying techniques are increasingly being considered for commercial applications, since low temperature and low oxygen environment reduce heat-induced and oxidative damages of the material during processing. Therefore, the aim of this study was to compare the effect of three different drying techniques (air-drying, freeze-drying and CO2 drying) on the bioactive potential of basil, and to determine the optimal processing conditions that provide the highest level of basil's native bioactive compounds. The contents of total phenols, total flavonoids and total chlorophylls (chlorophyll a and chlorophyll b), as well as the antioxidant capacity, using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radical scavenging assays, were determined by UV/Vis spectrophotometric methods. Phenolic acids in fresh and dried basil samples were identified and quantified using reversed phase high performance liquid chromatography (HPLC-PDA). Total phenol and flavonoid contents of basil samples varied between 10.55 – 35.01 mg GAE/g DM and from 4.30 to 18.70 mg GAE/g DM, respectively. The antioxidant capacity of basil samples ranged from 45.69 to 207.59 µmol Trolox/g DM (determined with DPPH assay) and from 75.48 to 215.66 µmol Trolox/g DM (determined with ABTS assay). Among the 4 identified phenolic acids (caftaric, caffeic, chicoric and rosmarinic acid), rosmarinic acid was the most abundant in all analyzed samples. According to the obtained results, freeze drying was recognized as the most suitable technique for preservation of polyphenolic compounds, providing the highest levels of bioactive compounds and antioxidant capacity in dried basil samples, with values closely comparable to the ones obtained for fresh basil. Regarding the CO2 drying technique, the combination of 40 °C during 2 hours, and 80 and 100 bars, were found to be the most efficient drying conditions for maintaining the native bioactive composition of basil (in accordance with the one obtained for the freeze dried sample). Air drying of basil resulted with the lowest content of phenolic compounds and chlorophylls. Taking into account that freeze drying is rather expensive process, the obtained results imply the need for further development and optimization of CO2 drying technique in this field, especially considering the high quality of final dried product.

Upon modern consumers’ demands for higher intake of bioactive/functional compounds in daily diet, consumption of Camellia sinensis products, including tea, arises, due to its pleasant aroma and numerous health-promoting properties, attributed to the presence of phytochemicals, primarily catechins and catechin-gallates as representatives of polyphenols, and caffeine among methylxanthines. There is currently an increasing interest in innovative extraction techniques such as Microwave-Assisted Extraction (MAE), Ultrasonic-Assisted Extraction (UAE) and High Hydrostatic Pressure (HHP) that enable fast and efficient extraction of phytochemicals as functional ingredients from various plant matrixes. As opposed to previously employed techniques for the extraction of polyphenols from tea, HHP extraction can be operated at room temperature, avoiding the use of high temperatures that can deteriorate thermo-sensitive ingredients and result with loss of their biological activities. Therefore, the aim of this study was to compare the extraction efficiency of polyphenols and methylxanthines from four different types of teas (white, green, oolong and black teas), by employing conventional and HHP extraction using water and hydro-alcoholic mixture as the extraction solvents. The content of total polyphenols was determined spectrophotometrically, individual polyphenols and methylxanthines were determined by means of high performance liquid chromatography (HPLC) analysis, while 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assays were used to assess the antioxidant capacity of tea extracts. Green tea generally exhibited the highest content of total and individual polyphenolic compounds, as well as the antioxidant capacity, among all investigated teas. The combination of conventional extraction using 50% ethanol (v/v) as the extraction solvent at 100°C and stirring for 10 min was the most efficient for extraction of polyphenols from teas. Flavan-3-ols were the prevalent group of polyphenols, with epigallocatechin gallate being the most abundant compound (ranging from 43.14 to 2885.50 mg/L), followed by caffeine (ranging from 98.27 to 1895.97 mg/L) among methylxanthines. In comparison to conventional extraction, high hydrostatic pressure provided lower extraction yield of bioactive compounds, regardless of the type of tea or extraction solvent, indicating that the use of higher temperature is the determining parameter for the extraction of bioactive compounds from teas.

The quality of chocolate is influenced by numerous factors, but the main characteristics that determine its consumer acceptability comprise chocolate’s rheological behaviour, textural properties, melting characteristics, sensory perception and lately the content of bioactive compounds, attributed for the numerous evidenced health benefits of chocolate. With the increase of dark chocolate and speciality niche products in the modern chocolate confectionery industry, a better understanding of these factors could have significant commercial implications. In the present study the effect of cocoa parts content (29, 38, 60, 72 and 85%) on the textural, bioactive and sensory properties of chocolate was evaluated. The content of polyphenolic compounds, as well as the antioxidant capacity were determined spectrophotometrically, while the textural analysis was conducted by employing probes that simulate the processes taking place in the mouth during mastication and chewing, and included penetration probe ; three point bending test and Kramer shear cell. Consumer evaluation of sensory properties was conducted using hedonic scale preference analysis. The cocoa parts content affected the polyphenolic compounds and antioxidant capacity, as well as textural properties, in the same way. Higher cocoa parts content is accounted for a higher polpyhenolic content and consequently higher antioxidant capacity, amounting to 11.5 mg GAE/g of total polyphenols in dark chocolate (85% cocoa parts). Similarly, higher cocoa parts content results with higher initial hardness during penetration, bending of chocolates and simulated mastication. The force required for penetration ranged from 22-26N (29% cocoa parts) up to maximal values of 42-45N (85% cocoa parts), which was confirmed during bending test that required higher force to break the chocolate bar with higher cocoa parts content. An inverse relation between the cocoa parts content and the sensory properties of chocolates was established. Chocolates containing lower cocoa parts content (29% and 38%) were preferred over the ones with a higher cocoa parts content, which might be attributed to higher sugar content of milk chocolate (29% cocoa parts) and pronounced bitterness and astringency attributes of dark chocolates (72% and 85% cocoa parts).

Consumption of various alcoholic beverages in form of plain and aromatized spirits and liqueurs is an important part of local and traditional gastronomy throughout the world. Although in the Southeast European and Mediterranean regions, herbal spirits and liqueurs are most commonly consumed, various cocoa-based and chocolate liqueurs can beside providing attractive taste and aroma, contribute to intake of a significant amount of cocoa-derived phytochemicals, especially polyphenols and methylxanthines which exhibit high antioxidant potential. Since the scientific information about the bioactive potential and the content of beneficial antioxidants which can be consumed by intake of cocoa-based alcoholic beverages are limited, chocolate liqueurs containing 20% of ethanol (alcohol content) and four types of chocolates differing in their cocoa parts (30%, 32%, 50% and 72%) were evaluated in this study. As stability of bioactive compounds and antioxidant capacity can be affected during processing and storage, which can influence the consumer`s requirements for high quality of final product, the stability of bioactive potential of chocolate liqueurs during 6 months of storage was examined. In order to compare the composition of polyphenols and methylxanthines of different chocolate liqueurs, UV/VIS spectrophotometric (total polyphenols and flavonoids, flavan-3-ols and proanthocyanidins) and high-performance liquid chromatography (HPLC-PDA) methods were applied. Antioxidant capacity was evaluated using DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS (2, 2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) assays. The content of bioactive compounds and antioxidant potential of chocolate liqueurs were significantly affected by the length of storage, exhibiting a decreasing tendency by prolongation of storage period. Chocolate liqueur consisting of dark chocolate with 72% of cocoa content exhibited the highest bioactive potential, in terms of phytochemical content and antioxidant capacity, confirming that the content of polyphenols and methylxanthines, as well as the antioxidant capacity of cocoa products depend on the cocoa content. Theobromine was the most abundant bioactive compound in chocolate liqueurs displayed by HPLC analysis, followed by (-) – epicatechin as the representative polyphenolic compound, which were determined in markedly higher contents in liqueur made of dark chocolate (72% cocoa parts). The obtained results might serve as a good starting point for production of a novel functional beverage, but with need for further investigations.

Extraction and product recovery are the most imperative steps in evaluation of valuable biologically active compounds from different plant substrates and ensuring availability of high-quality herbal products to consumers worldwide. With the growing interest in phytochemicals that exhibit positive health effects, for use in the production of functional food products, a need arises for the development of an efficient, simple, selective, environmentally friendly and economically feasible extraction technique, which will provide extracts prepared with non-toxic solvents. As the result of growing interest in innovative extraction techniques for the extraction of bioactive compounds, conventional extraction techniques that use water as the extraction media, such as infusion, decoction and maceration have been neglegted in recent years. Innovative extraction techniques including Microwave-Assisted Extraction (MAE) and Ultrasonic-Assisted Extraction (UAE) are much faster and efficient, and have significant advantages over conventional methods in terms of reduction in organic solvent consumption, process duration, minimizing sample degradation and elimination of undesirable and insoluble components from the extract. In this study the extraction efficiency of polyphenols from three traditional, widely used medicinal plants ; nettle (Urtica dioica L.), hawthorn (Crateagus oxycantha L.) and olive leaf (Olea europaea L.) by employing conventional and innovative extraction techniques was evaluated. In order to obtain an extract for use in food purposes, the application of organic solvents for the extraction of polyphenols was avoided by using water as the extraction solvent. Three conventional (infusions, maceration and decoction) and one innovative extraction technique (Ultrasonic-Assisted Extraction (UAE)) of medicinal plants, as well as their combination were employed. Bioactive potential of the obtained extracts was evaluated in terms of their polyphenolic profile and antioxidant properties by using spectrophotometric methods and high performance liquid chromatography (HPLC). According to the obtained results, among conventional extraction techniques, infusions of all three medicinal plants provided the highest and macerates the lowest polyphenolic yield, while the high temperature maintained during preparation of decoctions led to degradation of polyphenols when compared to infusions. Ultrasonically prepared extracts were more abundant in polyphenols than conventinally prepared extracts. The combination of ultrasonic-treatment prior to maceration and infusion revealed to be highly efficient for the extraction of polyphenols from all medicinal plants. The highest extraction efficiency of polyphenols was observed in ultrasound-pretreated and infused extracts. The extraction efficiency of specific polyphenols from different medicinal plants was dependant on the employed extraction technique, as well as the antioxidant properties of extracts which did not correlate well with total phenols, thus implying on the selective effect of employed techniques.