Performance of ß-carotene-loaded nanostructured lipid carriers under dynamic in vitro digestion system: Influence of the emulsifier type.

A better understanding of how emulsifier type could differently influence the behavior of nanostructured lipid carriers (NLC) under the gastrointestinal digestion process, as well as at the cellular level, is of utmost importance for the NLC-based formulations' optimization and risk assessment in the food field. In this study, NLC composed by fully hydrogenated soybean and high-oleic sunflower oils were prepared using soy lecithin (NLC Lß) or Tween 80 (NLC Tß) as an emulsifier. ß-Carotene was entrapped within NLC developed as a promising strategy to overcome ß-carotene's low bioavailability and stability. The effect of emulsifier type on the digestibility of ß-carotene-loaded NLC was evaluated using an in vitro dynamic digestion model mimicking peristalsis motion. The influence of ß-carotene-loaded NLC on cell viability was assessed using Caco-2 cells in vitro. NLC Tß remained stable in the gastric compartment, presenting particle size (PS) similar to the initial NLC (PS: 245.68 and 218.18 nm, respectively), while NLC Lß showed lower stability (PS > 1000 nm) in stomach and duodenum phases. NLC Tß also provided high ß-carotene protection and delivery capacity (i.e., ß-carotene bioaccessibility increased 10-fold). Based on the results of digestion studies, NLC Tß has shown better physical stability during the passage through the in vitro dynamic gastrointestinal system than NLC Lß. Moreover, the developed NLC did not compromise cell viability up to 25 µg/mL of ß-carotene. Thus, the NLC developed proved to be a biocompatible structure and able to incorporate and protect ß-carotene for further food applications. PRACTICAL APPLICATION: The findings of this study hold significant implications for industrial applications in terms of developing nanostructured lipid carriers from natural raw materials widely available and used to produce other lipid-based products in the food industry, as an alternative to synthetic ones. In this respect, the ß-carotene-loaded NLC developed in this study would find a great industrial application in the food industry, which is in constant search to develop functional foods capable of increasing the bioavailability of bioactive compounds.

Incorporation of curcumin-loaded solid lipid nanoparticles into yogurt: Tribo-rheological properties and dynamic in vitro digestion.

The incorporation of nanostructures loaded with bioactive compounds into food matrices is a promising approach to develop new functional foods with improved nutritional, health profiles and good sensorial properties. The rheological and tribological properties of yogurt enriched with curcumin-loaded solid lipid nanoparticles (SLN) were evaluated. Also, the TCA solubility index, the bioaccessibility of curcumin and cell viability were assessed after dynamic in vitro digestion. The presence of SLN in yogurt did not affect its rheological properties; however, SLN addition increased the lubrication capability of yogurt. After in vitro digestion, yogurt with added SLN (yogurt_SLN) presented a lower TCA solubility index (22 %) than the plain yogurt (39 %). The bioaccessibility and stability of curcumin were statistically similar for yogurt_SLN (30 % and 42 %, respectively) and SLN alone (20 % and 39 %, respectively). Regarding cell viability results, the intestinal digesta filtrates of both controls (i.e., SLN alone and plain yogurt) did not affect significantly the cell viability, while the yogurt_SLN presented a possible cytotoxic effect at the concentrations tested. In general, the incorporation of SLN into yogurt seemed to promote the mouthfeel of the yogurt and did not adversely affect the bioaccessibility of curcumin. However, the interaction of SLN and yogurt matrix seemed to have a cytotoxic effect after in vitro digestion, which should be further investigated. Despite that, SLN has a high potential to be used as nanostructure in a functional food as a strategy to increase the bioactive compounds' bioaccessibility.

Development and characterization of nanostructured lipid carriers for cannabidiol delivery.

This study evaluated the physicochemical characteristics of nanostructured lipid carriers (NLCs) as a potential vehicle for cannabidiol (CBD), a lipophilic molecule with great potential to promote health benefits. NLCs were produced using hemp seed oil and fully-hydrogenated soybean oil at different proportions. The emulsifiers evaluated were soybean lecithin (SL), Tween 80 (T80) and a mixture of SL:T80 (50:50). CBD was tested in the form of CBD-rich extract or isolate CBD, to verify if it affects the NLCs characteristics. Based on particle size and polydispersity, SL was considered the most suitable emulsifier to produce the NLCs. All lipid proportions evaluated had no remarkable effect on the physicochemical characteristics of NLCs, resulting in CBD-loaded NLCs with particle size below 250 nm, high CBD entrapment efficiency and CBD retention rate of 100% for 30 days, demonstrating that NLCs are a suitable vehicle for both CBD-rich extract or isolate CBD.

Incorporation of curcumin-loaded lipid-based nano delivery systems into food: Release behavior in food simulants and a case study of application in a beverage.

Curcumin-loaded lipid-based nano delivery systems (nanoemulsions-NE, solid lipid nanoparticles-SLN and nanostructured lipid carriers-NLC) were subjected to different food simulants to evaluate curcumin's in vitro release kinetics and particlés stability. The nano delivery systems were also incorporated into a model beverage and their physicochemical properties were evaluated during storing period. Curcumin's bioaccessibility of beverages containing nano delivery systems were assessed through an in vitro digestion process. All nano delivery systems showed a higher curcumin's release and lower particle stability at 50 % ethanol (lipophilic food simulant) comparatively to hydrophilic food simulants. NLC and SLN showed a good particle's stability within the beverage during storing period, while NE presented high instability immediately after incorporation in the beverage. NLC and SLN did not affect beverage's stability relatively to pH, however the beverage with NLC was slightly more stable regarding color. Beverage with SLN presented higher curcumin bioaccessibility compared to the beverage with NLC, however it showed lower curcumin's stability.

An in vitro fermentation model to study the impact of bacteriophages targeting Shiga toxin-encoding Escherichia coli on the colonic microbiota.

Lytic bacteriophages are considered safe for human consumption as biocontrol agents against foodborne pathogens, in particular in ready-to-eat foodstuffs. Phages could, however, evolve to infect different hosts when passing through the gastrointestinal tract (GIT). This underlines the importance of understanding the impact of phages towards colonic microbiota, particularly towards bacterial families usually found in the colon such as the Enterobacteriaceae. Here we propose in vitro batch fermentation as model for initial safety screening of lytic phages targeting Shiga toxin-producing Escherichia coli (STEC). As inoculum we used faecal material of three healthy donors. To assess phage safety, we monitored fermentation parameters, including short chain fatty acid production and gas production/intake by colonic microbiota. We performed shotgun metagenomic analysis to evaluate the outcome of phage interference with colonic microbiota composition and functional potential. During the 24 h incubation, concentrations of phage and its host were also evaluated. We found the phage used in this study, named E. coli phage vB_EcoS_Ace (Ace), to be safe towards human colonic microbiota, independently of the donors' faecal content used. This suggests that individuality of donor faecal microbiota did not interfere with phage effect on the fermentations. However, the model revealed that the attenuated STEC strain used as phage host perturbed the faecal microbiota as based on metagenomic analysis, with potential differences in metabolic output. We conclude that the in vitro batch fermentation model used in this study is a reliable safety screening for lytic phages intended to be used as biocontrol agents.

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori.

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.

Emerging challenges in assessing bio-based nanosystems' behaviour under in vitro digestion focused on food applications - A critical view and future perspectives.

The current consumers' demand for high quality food products together with the growing awareness regarding the link between health and nutrition has led to the development of novel food products with added functionality. Such functionality can be modulated by adding bio-based nanosystems that can improve the bioaccessibility of bioactive compounds and facilitate nutrient absorption. However, these functional properties can be significantly affected by the adverse conditions (e.g., low pH, presence of enzymes, salts) of the gastrointestinal tract. As such, understanding the behaviour of such delivery systems under digestion conditions is of utmost importance and several analytical tools and in vitro digestion models have been used for this purpose. This review summarizes the latest updates on nanosystems' performance under in vitro digestion and provides critical insights related to important and complementary analytical tools (e.g., rheology, Raman spectroscopy, x-ray scattering) used to assess their performance throughout digestion. Furthermore, the most prominent and frequent challenges associated with such in vitro analyses are also described, together with the current trends regarding the development of in vitro digestion models and some considerations that should be undertaken for their validation. Efforts must be made towards developing reliable and standard in vitro digestion models that use sophisticated analytical techniques to further expand the knowledge regarding nanosystems' behaviour under in vitro digestion conditions.

Incorporation of Solid Lipid Nanoparticles into Stirred Yogurt: Effects in Physicochemical and Rheological Properties during Shelf-Life.

The aim of this work was to develop a yogurt fortified with curcumin. Curcumin is a lipophilic compound with a wide range of biological activities; however, it presents low water solubility and low bioavailability, and therefore it was the first to be encapsulated in solid lipid nanoparticles (SLNs). Then the influence of the incorporation of curcumin-loaded SLNs on the physicochemical (i.e., pH, titratable acidity, syneresis and color) and rheological properties of yogurt during its shelf-life (30 days at 4 °C) was evaluated. SLN incorporation into yogurt did not affect pH and titratable acidity compared to the control (i.e., plain yogurt) during shelf-life, even though the yogurt with SLNs presented lower values of pH (4.25 and 4.34) and acidity (0.74% lactic acid and 0.84% lactic acid) than the control in the end, respectively. Furthermore, the yogurt with SLNs presented slightly higher values of syneresis than the control during the shelf-life; however, it did not present visual differences in whey separation. Relative to the color, the incorporation of SLNs into the yogurt imparted a strong yellow color to the sample but did not affect color stability during shelf-life. Both samples showed flow curves with yield stress and shear-thinning behavior during shelf-life, and, regarding the viscoelastic behavior, both showed a typical weak viscoelastic gel with an elastic structure. Overall, curcumin-loaded SLNs incorporation did not affect the physicochemical and rheological stability of yogurt during shelf-life, showing a promising application for the development of new functional foods.

Lipid-based nanostructures as a strategy to enhance curcumin bioaccessibility: Behavior under digestion and cytotoxicity assessment.

The aim of this study was to evaluate the behavior of different lipid-based nanostructures during in vitro digestion, in particular on curcumin's bioaccessibility, and to access their potential toxicity. Solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions (NE) were submitted to harmonized static in vitro digestion and their cytotoxicity and cellular transport were evaluated using Caco-2 cell line. NE presented the highest curcumin's bioaccessibility followed by NLC and SLN, 71.1%, 63.7% and 53.3%, respectively. Free fatty acids percentage increased in the following order: NLC ≤ NE < SLN. Non-digested nanostructures and excipients presented no cytotoxicity; however, digested NE and NLC presented cytotoxicity due to MCT oil, which presented cytotoxicity after digestion. The apparent permeability coefficient of NLC was higher than SLN and NE. These results showed that lipid-based nanostructures' physical state and composition have a high influence on particles' behavior during digestion, and on their cytotoxicity/intestinal permeability, and highlights the importance of conducting cytotoxicity assessments after in vitro digestion. This work contributes to a better understanding of the behavior of lipid-based nanostructures under digestion/adsorption, and this knowledge will be useful in design of nanostructures that afford both safety and an increased bioactive compounds' bioavailability.

Nanoemulsions for Enhancement of Curcumin Bioavailability and Their Safety Evaluation: Effect of Emulsifier Type.

This work aimed at evaluating the effects of different emulsifiers on curcumin-loaded nanoemulsions' behavior during digestion, its safety and absorption, to develop nanoemulsions that provide safety and improved curcumin functionality. Nanoemulsions (NEs) were produced using two bio-based (lecithin (LEC) and rhamnolipids (RHAM)) and one synthetic (Tween®80 (TWE)) emulsifier at similar concentrations. Different NEs were subjected to in vitro digestion. The cytotoxicity and permeability tests were performed in Caco-2 cells. NE_TWE were stable during all phases of in vitro digestion, whereas NE_LEC and NE_RHAM were found to be unstable from the gastric phase. NE_TWE showed 100% of free fatty acids released, followed by NE_RHAM and NE_LEC. Curcumin's bioaccessibility and stability increased in the following order: NE_LEC > NE_RHAM > NE_TWE. NE_LEC and NE_TWE did not show cytotoxic effects in any of the concentrations tested, while NE_RHAM presented high cytotoxicity in all concentrations tested. The apparent permeability coefficients were determined for NE_LEC and NE_TWE; however, the results were not statistically different. These results showed that the emulsifier used has a high impact on nanoemulsions' behavior under the digestion process and on their cytotoxicity. This work contributed to the state-of-the-art's progress on the development of safer curcumin delivery systems with improved functionality, particularly regarding the proper selection of ingredients to produce said systems.

Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610.

The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4 to 6 was obtained, presenting high stability after a static in vitro digestion (98.5 ± 0.2%). The maximum production yield expressed as total XOS per amount of xylan (306 ± 4 mg/g) was achieved after 8 h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5 °C, using 2.5 g/L of initial concentration of xylan increased up to 5.0 g/L at 3 h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield.