Application of inulin for the formulation and delivery of bioactive molecules and live cells.

Inulin is a fructan biosynthesized mainly in plants of the Asteraceae family. It is also found in edible vegetables and fruits such as onion, garlic, leek, and banana. For the industrial production of inulin, chicory and Jerusalem artichoke are the main raw material. Inulin is used in the food, pharmaceutical, cosmetic as well biotechnological industries. It has a GRAS status and exhibits prebiotic properties. Inulin can be used as a wall material in the encapsulation process of drugs and other bioactive compounds and the development of their delivery systems. In the review, the use of inulin for the encapsulation of probiotics, essential and fatty oils, antioxidant compounds, natural colorant and other bioactive compounds is presented. The encapsulation techniques, materials and the properties of final products suitable for the delivery into food are discussed. Research limitations are also highlighted.

Kinetic Study of Encapsulated ß-Carotene Degradation in Aqueous Environments: A Review.

The provitamin A activity of ß-carotene is of primary interest to address one of the world's major malnutrition concerns. ß carotene is a fat-soluble compound and its bioavailability from natural sources is very poor. Hence, studies have been focused on the development of specific core/shell micro- or nano-structures that encapsulate ß-carotene in order to allow its dispersion in liquid systems and improve its bioavailability. One key objective when developing these structures is also to accomplish ß-carotene stability. The aim of this review is to collect kinetic data (rate constants, activation energy) on the degradation of encapsulated ß-carotene in order to derive knowledge on the possibility for these systems to be scaled-up to the industrial production of functional foods. Results showed that most of the nano- and micro-structures designed for ß-carotene encapsulation and dispersion in the water phase provide better protection with respect to a natural matrix, such as carrot juice, increasing the ß-carotene half-life from about 30 d to more than 100 d at room temperature. One promising approach to increase ß-carotene stability was found to be the use of wall material, surfactants, or co-encapsulated compounds with antioxidant activity. Moreover, a successful approach was the design of structures, where the core is partially or fully solidified; alternatively, either the core or the interface or the outer phase are gelled. The data collected could serve as a basis for the rational design of structures for ß-carotene encapsulation, where new ingredients, especially the extraordinary natural array of hydrocolloids, are applied.

Kinetic Study of Encapsulated ß-Carotene Degradation in Dried Systems: A Review.

ß-Carotene serves as a precursor of vitamin A and provides relevant health benefits. To overcome the low bioavailability of ß-carotene from natural sources, technologies have been designed for its encapsulation in micro- and nano-structures followed by freeze-drying, spray-drying, supercritical fluid-enhanced dispersion and electrospraying. A technological challenge is also to increase ß-carotene stability, since due to its multiple conjugated double bonds, it is particularly prone to oxidation. This review analyzes the stability of ß-carotene encapsulated in different dried micro- and nano-structures by comparing rate constants and activation energies of degradation. The complex effect of water activity and glass transition temperature on degradation kinetics is also addressed, since the oxidation process is remarkably dependent on the glassy or collapsed state of the matrix. The approaches to improve ß-carotene stability, such as the development of inclusion complexes, the improvement of the performance of the interface between air and oil phase in which ß-carotene was dissolved by application of biopolymer combinations or functionalization of natural biopolymers, the addition of hydrophilic small molecular weight molecules that reduce air entrapped in the powder and the co-encapsulation of antioxidants of various polarities are discussed and compared, in order to provide a rational basis for further development of the encapsulation technologies.

An Optimization Procedure for Preparing Aqueous CAR/HP-CD Aggregate Dispersions.

ß-Carotene is a very important molecule for human health. It finds a large application in the food industry, especially for the development of functional foods and dietary supplements. However, ß-carotene is an unstable compound and is sensitive to light, temperature, and oxygen. To overcome those limitations, various delivery systems were developed. The inclusion of ß-carotene by cyclodextrin aggregates is attractive due to non-toxicity, low hygroscopicity, stability, and the inexpensiveness of cyclodextrins. In this study, ß-carotene/2-hydroxypropyl-ß-cyclodextrin aggregates were prepared based on the procedure of the addition of ß-carotene in an organic solvent to the hot water dispersion of 2-hydroxypropyl-ß-cyclodextrin and the following instant evaporation of the organic solvent. The best conditions for the aggregate preparation were found to be as follows: 25% concentration of 2-hydroxypropyl-ß-cyclodextrin in water, 65 °C temperature, and acetone for ß-carotene dissolution. The efficiency of entrapping was equal to 88%. The procedure is attractive due to the short time of the aggregate preparation.

Microencapsulation of Bioactive Ingredients for Their Delivery into Fermented Milk Products: A Review.

The popularity and consumption of fermented milk products are growing. On the other hand, consumers are interested in health-promoting and functional foods. Fermented milk products are an excellent matrix for the incorporation of bioactive ingredients, making them functional foods. To overcome the instability or low solubility of many bioactive ingredients under various environmental conditions, the encapsulation approach was developed. This review analyzes the fortification of three fermented milk products, i.e., yogurt, cheese, and kefir with bioactive ingredients. The encapsulation methods and techniques alongside the encapsulant materials for carotenoids, phenolic compounds, omega-3, probiotics, and other micronutrients are discussed. The effect of encapsulation on the properties of bioactive ingredients themselves and on textural and sensory properties of fermented milk products is also presented.

Nisin-Loaded Ulvan Particles: Preparation and Characterization.

Nisin is an attractive alternative to chemical preservatives in the food industry. It is a cationic peptide of 34 amino acid residues that exhibits antimicrobial activity against Gram-positive bacteria. To ensure nisin stability in food matrices, new nisin-loaded ulvan particles were developed by the complexation method. The interaction of nisin with ulvan was demonstrated by FT-IR spectroscopy and differential scanning calorimetry. The encapsulation efficiency was calculated at different pH values within the range of 4.0-7.0 and was found to have the highest value at pH 7.0. The size and surface charge of particles fabricated at different concentrations of nisin and pH values were determined. Nisin-loaded ulvan particles exhibited antimicrobial activity against Gram-positive bacteria comparable to that of free nisin. Therefore, the developed complexes have the potential for application as biopreservatives in the food industry. For the first time, the potential of ulvan as a carrier of antimicrobial agent nisin was demonstrated.

European Database of Carotenoid Levels in Foods. Factors Affecting Carotenoid Content.

Many studies indicate that diets including carotenoid-rich foods have positive effects on human health. Some of these compounds are precursors of the essential nutrient vitamin A. The present work is aimed at implementing a database of carotenoid contents of foods available in the European market. Factors affecting carotenoid content were also discussed. Analytical data available in peer-reviewed scientific literature from 1990 to 2018 and obtained by HPLC/UHPLC were considered. The database includes foods classified according to the FoodEx2 system and will benefit compilers, nutritionists and other professionals in areas related to food and human health. The results show the importance of food characterization to ensure its intercomparability, as large variations in carotenoid levels are observed between species and among varieties/cultivars/landraces. This highlights the significance of integrating nutritional criteria into agricultural choices and of promoting biodiversity. The uncertainty quantification associated with the measurements of the carotenoid content was very rarely evaluated in the literature consulted. According to the EuroFIR data quality evaluation system for food composition tables, the total data quality index mean was 24 in 35, reflecting efforts by researchers in the analytical methods, and less resources in the sampling plan documentation.

Inactivation of Bacteria Using Bioactive Nanoparticles and Alternating Magnetic Fields.

Foodborne pathogens are frequently associated with risks and outbreaks of many diseases; therefore, food safety and processing remain a priority to control and minimize these risks. In this work, nisin-loaded magnetic nanoparticles were used and activated by alternating 10 and 125 mT (peak to peak) magnetic fields (AMFs) for biocontrol of bacteria Listeria innocua, a suitable model to study the inactivation of common foodborne pathogen L. monocytogenes. It was shown that L. innocua features high resistance to nisin-based bioactive nanoparticles, however, application of AMFs (15 and 30 min exposure) significantly potentiates the treatment resulting in considerable log reduction of viable cells. The morphological changes and the resulting cellular damage, which was induced by the synergistic treatment, was confirmed using scanning electron microscopy. The thermal effects were also estimated in the study. The results are useful for the development of new methods for treatment of the drug-resistant foodborne pathogens to minimize the risks of invasive infections. The proposed methodology is a contactless alternative to the currently established pulsed-electric field-based treatment in food processing.

Carotenoids: Considerations for Their Use in Functional Foods, Nutraceuticals, Nutricosmetics, Supplements, Botanicals, and Novel Foods in the Context of Sustainability, Circular Economy, and Climate Change.

Carotenoids are versatile isoprenoids that are important in food quality and health promotion. There is a need to establish recommended dietary intakes/nutritional reference values for carotenoids. Research on carotenoids in agro-food and health is being propelled by the two multidisciplinary international networks, the Ibero-American Network for the Study of Carotenoids as Functional Foods Ingredients (IBERCAROT; http://www.cyted.org) and the European Network to Advance Carotenoid Research and Applications in Agro-Food and Health (EUROCAROTEN; http://www.eurocaroten.eu). In this review, considerations for their safe and sustainable use in products mostly intended for health promotion are provided. Specifically, information about sources, intakes, and factors affecting bioavailability is summarized. Furthermore, their health-promoting actions and importance in public health in relation to the contribution of reducing the risk of diverse ailments are synthesized. Definitions and regulatory and safety information for carotenoid-containing products are provided. Lastly, recent trends in research in the context of sustainable healthy diets are summarized.

New ß-Carotene-Chitooligosaccharides Complexes for Food Fortification: Stability Study.

The application of ß-carotene in food industry is limited due to its chemical instability. The drawback may be overcome by designing new delivery systems. The stability of ß-carotene complexed with chitooligosaccharides by kneading, freeze-drying and sonication methods was investigated under various conditions. The first-order kinetics parameters of the reaction of ß-carotene degradation were calculated. The complexation improved the stability of ß-carotene at high temperatures and ensured its long-term stability in the dark at 4 °C and 24 °C, and in the light at 24 °C. In water solutions, the best characteristics were exhibited by the complexes prepared by freeze-drying and sonication methods. In the powder form, the complexes retained their colour for the period of the investigation of four months. The calculated total colour differences of the complexes were qualified as appreciable, detectable by ordinary people, but not large. Therefore, ß-carotene-chitooligosaccharides complexes could be used as a new delivery system suitable for food fortification.

Preparation and characterisation of novel water-soluble ß-carotene-chitooligosaccharides complexes.

ß-carotene and chitooligosaccharides are bioactive compounds that find their application in the food industry as well in biomedical fields. However, the application of ß-carotene is limited due to its very low water solubility, as well as its air, light and temperature sensitivity. The preparation of ß-carotene-chitooligosaccharides complexes by mechanochemical methods was presented. Their physical and chemical properties including solubility, size, zeta potential and radical scavenging activity were investigated. The interaction of the two components was shown by NMR, FT-IR, and Raman spectroscopy. The complexes were analysed by scanning and transmission electron microscopy. Chitooligosaccharides could serve as a carrier for ß-carotene delivery. The complexation did not cause the loss of the radical scavenging activity of ß-carotene and guaranteed its water solubility.

Preparation and characterization of iron oxide magnetic nanoparticles functionalized by nisin.

Nisin is a known bacteriocin approved as a food additive for food preservation. It exhibits a wide spectrum antimicrobial activity against Gram-positive bacteria. Iron oxide magnetic nanoparticles were synthesized and characterized by X-ray diffraction method. A main part of iron oxide nanoparticles was found to be maghemite though a small quantity of magnetite could also be present. Magnetic nanoparticles were stabilized by citric, ascorbic, gallic or glucuronic acid coating. Stable iron oxide magnetic nanoparticles were functionalized by nisin using a simple and low cost adsorption method. Nisin loading was confirmed by FT-IR spectra, thermogravimetric analysis, dynamic light scattering and atomic force microscopy methods. Nisin-loaded iron oxide magnetic nanoparticles were stable at least six weeks as judged by the measurements of zeta-potential and hydrodynamic diameter. The antimicrobial activity of nisin-loaded iron oxide magnetic nanoparticles was demonstrated toward Gram-positive bacteria. Functionalized nanoparticles could therefore find the application as antimicrobials in innovative and emerging technologies based on the magnetic field.

Origin and pathophysiology of protein carbonylation, nitration and chlorination in age-related brain diseases and aging.

Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.

Inactivation of Escherichia coli Using Nanosecond Electric Fields and Nisin Nanoparticles: A Kinetics Study.

Nisin is a recognized bacteriocin widely used in food processing, however, being ineffective against gram-negative bacteria and in complex food systems. As a result, the research of methods that have cell wall-permeabilizing activity is required. In this study, electroporation to trigger sensitization of gram-negative bacteria to nisin-loaded pectin nanoparticles was used. As a model microorganism, bioluminescent strain of E. coli was introduced. Inactivation kinetics using nanosecond pulsed electric fields (PEFs) and nisin nanoparticles have been studied in a broad range (100-900 ns, 10-30 kV/cm) of pulse parameters. As a reference, the microsecond range protocols (100 µs × 8) have been applied. It was determined that the 20-30 kV/cm electric field with pulse duration ranging from 500 to 900 ns was sufficient to cause significant permeabilization of E. coli to trigger a synergistic response with the nisin treatment. The kinetics of the inactivation was studied with a time resolution of 2.5 min, which provided experimental evidence that the efficacy of nisin-based treatment can be effectively controlled in time using PEF. The results and the proposed methodology for rapid detection of bacteria inactivation rate based on bioluminescence may be useful in the development and optimization of protocols for PEF-based treatments.

Impact of pectin esterification on the antimicrobial activity of nisin-loaded pectin particles.

The relationship between pectin structure and the antimicrobial activity of nisin-loaded pectin particles was examined. The antimicrobial activity of five different nisin-loaded pectin particles, i.e., nisin-loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0-7.0 by agar-diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin-loaded pectin particles. Nisin-loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin-loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin-loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245-251, 2017.

Overcoming Antimicrobial Resistance in Bacteria Using Bioactive Magnetic Nanoparticles and Pulsed Electromagnetic Fields.

Nisin is a known bacteriocin, which exhibits a wide spectrum of antimicrobial activity, while commonly being inefficient against Gram-negative bacteria. In this work, we present a proof of concept of novel antimicrobial methodology using targeted magnetic nisin-loaded nano-carriers [iron oxide nanoparticles (NPs) (11-13 nm) capped with citric, ascorbic, and gallic acids], which are activated by high pulsed electric and electromagnetic fields allowing to overcome the nisin-resistance of bacteria. As a cell model the Gram-positive bacteria Bacillus subtilis and Gram-negative Escherichia coli were used. We have applied 10 and 30 kV cm-1 electric field pulses (100 µs × 8) separately and in combination with two pulsed magnetic field protocols: (1) high dB/dt 3.3 T × 50 and (2) 10 mT, 100 kHz, 2 min protocol to induce additional permeabilization and local magnetic hyperthermia. We have shown that the high dB/dt pulsed magnetic fields increase the antimicrobial efficiency of nisin NPs similar to electroporation or magnetic hyperthermia methods and a synergistic treatment is also possible. The results of our work are promising for the development of new methods for treatment of the drug-resistant foodborne pathogens to minimize the risks of invasive infections.

Influence of L-homoarginine as an analogue of L-arginine on the heat-induced aggregation of proteins.

The influence of l-homoarginine on the heat-induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l-homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l-Homoarginine increased the onset temperature of heat-induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l-homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l-homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur.

High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

Influence of osmotic shock on Escherichia coli insoluble protein fraction in the presence of exogenous osmolytes.

The influence of osmotic shock in the presence of exogenous osmolytes on the formation and composition of insoluble protein fraction in Escherichia coli was investigated. Interferon-α5 (IFN-α5) expressed in E. coli BL21(DE3) was used as a model protein. Cells were cultivated at three different temperatures of 25, 30, and 37°C. Two different osmolytes were used. Glycine as an amino acid metabolized by E. coli, or betaine which is not metabolized by cells, was added to the growth medium in the presence of salt. In both cases (i.e. when metabolized or non-metabolized amino acid was used), IFN-α5 formed the aggregates, except at 25°C of cultivation. Moreover, the differences in the quantitative composition of insoluble protein fraction were revealed by the proteomic analysis. The amount of some identified proteins increased, decreased, or did not change in the samples cultivated under osmotic shock in the presence of betaine as compared with the sample cultivated without salt and betaine in growth medium.