A miniaturised semi-dynamic in-vitro model of human digestion.

Reliable in-vitro digestion models that are able to successfully replicate the conditions found in the human gastrointestinal tract are key to assess the fate and efficiency of new formulations aimed for oral consumption. However, current in-vitro models either lack the capability to replicate crucial dynamics of digestion or require large volumes of sample/reagents, which can be scarce when working with nanomaterials under development. Here, we propose a miniaturised digestion system, a digestion-chip, based on incubation chambers integrated on a polymethylmethacrylate device. The digestion-chip incorporates key dynamic features of human digestion, such as gradual acidification and gradual addition of enzymes and simulated fluids in the gastric phase, and controlled gastric emptying, while maintaining low complexity and using small volumes of sample and reagents. In addition, the new approach integrates real-time automated closed-loop control of two key parameters, pH and temperature, during the two main phases of digestion (gastric and intestinal) with an accuracy down to ± 0.1 °C and ± 0.2 pH points. The experimental results demonstrate that the digestion-chip successfully replicates the gold standard static digestion INFOGEST protocol and that the semi-dynamic digestion kinetics can be reliably fitted to a first kinetic order model. These devices can be easily adapted to dynamic features in an automated, sensorised, and inexpensive platform and will enable reliable, low-cost and efficient assessment of the bioaccessibility of new and expensive drugs, bioactive ingredients or nanoengineered materials aimed for oral consumption, thereby avoiding unnecessary animal testing.

Designing an antimicrobial film for wound applications incorporating bacteriophages and ε-poly-l-lysine.

Alginate-based dressings have been shown to promote wound healing, leveraging the unique properties of alginate. This work aimed to develop and characterize flexible individual and bilayered films to deliver bacteriophages (phages) and ε-Poly-l-lysine (ε-PLL). Films varied in different properties. The moisture content, swelling and solubility increased with higher alginate concentrations. The water vapour permeability, crucial in biomedical films to balance moisture levels for effective wound healing, reached optimal levels in bilayer films, indicating these will be able to sustain an ideal moist environment. The bilayer films showed improved ductility (lower tensile strength and increased elongation at break) compared to individual films. The incorporated phages maintained viability for 12 weeks under vacuum and refrigerated conditions, and their release was sustained and gradual. Antibacterial immersion tests showed that films with phages and ε-PLL significantly inhibited Pseudomonas aeruginosa PAO1 growth (>3.1 Log CFU/cm2). Particle release was influenced by the swelling degree and diffusional processes within the polymer network, providing insights into controlled release mechanisms for particles of varying size (50 nm to 6 µm) and charge. The films developed, demonstrated modulated release capabilities for active agents, and may show potential as controlled delivery systems for phages and wound healing adjuvants.

Active Low-Density Polyethylene-Based Films by Incorporating α-Tocopherol in the Free State and Loaded in PLA Nanoparticles: A Comparative Study.

In this work, alpha-tocopherol (α-TOC) was encapsulated in poly(lactic acid) nanoparticles (PLA NPs) and added to low-density polyethylene (LDPE) films with the aim of producing an active film for food packaging applications. PLA NPs loaded with α-TOC were produced through nanoprecipitation and dried using two methods (freeze-dryer and oven). LDPE-based films with final polymeric matrix concentrations of 10 and 20 g/kg were then produced through blow extrusion. The results showed that LDPE-based films loaded with α-TOC can be produced using blow extrusion, and a good distribution of PLA NPs can be obtained within the LDPE matrix as observed using scanning electron microscopy (SEM). The mechanical properties were affected by the incorporation of α-TOC and PLA NPs loaded with α-TOC, with the observation of a decrease in tensile strength and Young's Modulus values and an increase in elongation at break. Regarding water vapor permeability, the films showed a reduction in the values with the addition of α-TOC and PLA NPs loaded with α-TOC compared to the LDPE film (control). Films with α-TOC in the free state and loaded in PLA NPs showed antioxidant activity, but their behavior was affected by the encapsulation process.

Rhamnolipids: A biosurfactant for the development of lipid-based nanosystems for food applications.

Biosurfactants (surfactants synthesized by microorganisms) are produced by microorganisms and are suitable for use in different areas. Among biosurfactants, rhamnolipids are the most studied and popular, attracting scientists, and industries' interest. Due to their unique characteristics, the rhamnolipids have been used as synthetic surfactants' alternatives and explored in food applications. Besides the production challenges that need to be tackled to guarantee efficient production and low cost, their properties need to be adjusted to the final application, where the pH instability needs to be considered. Moreover, regulatory approval is needed to start being used in commercial applications. One characteristic of interest is their capacity to form oil-in-water nanosystems. Some of the most explored have been nanoemulsions, solid-lipid nanoparticles and nanostructured lipid carriers. This review presents an overview of the main properties of rhamnolipids, asserts the potential and efficiency of rhamnolipids to replace the synthetic surfactants in the development of nanosystems, and describes the rhamnolipids-based nanosystems used in food applications. It also discusses the main characteristics and methodologies used for their characterization and in the end, some of the main challenges are highlighted.

Resveratrol-loaded octenyl succinic anhydride modified starch emulsions and hydroxypropyl methylcellulose (HPMC) microparticles: Cytotoxicity and antioxidant bioactivity assessment after in vitro digestion.

Hydroxypropyl methylcellulose (HPMC)-based microparticles and modified starch emulsions (OSA-MS) were loaded with resveratrol and characterized regarding their physicochemical and thermal properties. Both delivery systems were subject to an in vitro gastrointestinal digestion to assess the bioaccessibility of resveratrol. In addition, cell-based studies were conducted after in vitro digestion and cytotoxicity and oxidative stress were assessed. HPMC-based microparticles displayed higher average sizes (d) and lower polydispersity index (PDI) (d = 948 nm, PDI < 0.2) when compared to OSA-MS-based emulsions (d = 217 nm, PDI < 0.3). Both proved to protect resveratrol under digestive conditions, leading to an increase in bioaccessibility. Resveratrol-loaded HPMC-microparticles showed a higher bioaccessibility (56.7 %) than resveratrol-loaded emulsions (19.7 %). Digested samples were tested in differentiated co-cultures of Caco-2 and HT29-MTX, aiming at assessing cytotoxicity and oxidative stress, and a lack of cytotoxicity was observed for all samples. Results displayed an increasing antioxidant activity, with 1.6-fold and 1.4-fold increases over the antioxidant activity of free resveratrol, for HPMC-microparticles and OSA-MS nanoemulsions, respectively. Our results offer insight into physiological relevancy due to assessment post-digestion and highlight the protection that the use of micro-nano delivery systems can confer to resveratrol and their potential to be used as functional food ingredients capable of providing antioxidant benefits upon consumption.

Recent advances in oral delivery systems of resveratrol: foreseeing their use in functional foods.

Herein, we review the current state-of-the-art on the use of micro- and nano-delivery systems, a possible solution to some of the drawbacks associated with the incorporation of resveratrol in foods. Specifically, we present an overview of a wide range of micro-nanostructures, namely, lipidic and polymeric, used for the delivery of resveratrol. Also, the gastrointestinal fate of resveratrol-loaded micro-nanostructures, as a critical parameter for their use as functional food, is explored in terms of stability, bioaccessibility, and bioavailability. Different micro-nanostructures are of interest for the development of functional foods given that they can provide different advantages and properties to these foods and even be tailor-made to address specific issues (e.g., controlled or targeted release). Therefore, we discuss a wide range of micro-nanostructures, namely, lipidic and polymeric, used to deliver resveratrol and aimed at the development of functional foods. It has been reported that the use of some production methodologies can be of greater interest than others, for example, emulsification, solvent displacement and electrohydrodynamic processing (EHDP) enable a greater increase in bioaccessibility. Additionally, the use of coatings facilitates further improvements in bioaccessibility, which is likely due to the increased gastric stability of the coated micro-nanostructures. Other properties, such as mucoadhesion, can also help improve bioaccessibility due to the increase in gut retention time. Additionally, cytotoxicity (e.g., biocompatibility, antioxidant, and anti-inflammatory) and possible sensorial impact of resveratrol-loaded micro- and nano-systems in foods are highlighted.

Characterization of Sodium Alginate-Based Films Blended with Olive Leaf and Laurel Leaf Extracts Obtained by Ultrasound-Assisted Technology.

Due to environmental concerns, there is an increasing need to reduce the use of synthetic and non-renewable packaging materials to reduce waste and increase sustainability. This study aimed to characterise sodium alginate edible-based films (SA) incorporated with laurel leaf extract (LLE) and olive leaf extract (OLE) obtained by ultrasound-assisted extraction. Determination of total phenolic content, antioxidant, and antimicrobial activity was performed for the extracts and films. Also, thickness, tensile strength, elongation at break, modulus of elasticity, opacity and colour, moisture content, water vapour permeability (WVP), Fourier-transform infrared spectroscopy (FTIR) spectra, and surface morphology by scanning electron microscope (SEM) analyses were performed for the films. LLE yielded better results in terms of phenolic content (195 mg GAE/g), antioxidant (2.1 TE/g extract) and antimicrobial activity (MIC at 1% for Listeria monocytogenes and Staphylococcus aureus, and 1.8% for Enterococcus faecalis). For the films, the simultaneous incorporation of LLE 1% (w/v) and OLE 1% (w/v) resulted in a significant reduction of approximately 2 log CFU/g against S. aureus. The addition of LLE and OLE extracts also proved to improve barrier properties (lower WVP for SA films with LLE 1% + OLE 1%, 3.49 × 10-11 g m-1 s-1 Pa-1) and promoted changes in resistance and flexibility. The results demonstrated that active alginate-based films can be valuable for enhancing food preservation.

Whey-pectin microcapsules improve the stability of grape marc phenolics during digestion.

Grape marc (GM) is an agri-food residue from the wine industry valuable for its high content of phenolic compounds. This study aimed to develop an encapsulation system for GM extract (GME) using food-grade biopolymers resistant to gastric conditions for its potential use as a nutraceutical. For this purpose, a hydroalcoholic GME was prepared with a total phenolics content of 219.62 ± 11.50 mg gallic acid equivalents (GAE)/g dry extract and 1389.71 ± 97.33 µmol Trolox equivalents/g dry extract antioxidant capacity, assessed through ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. Moreover, the extract effectively neutralized reactive oxygen species in Caco-2 cells, demonstrating an intracellular antioxidant capacity comparable to Trolox. The GME was encapsulated using whey protein isolate and pectin through nano spray drying (73% yield), resulting in spherical microparticles with an average size of 1 ± 0.5 µm and a polydispersity of 0.717. The encapsulation system protected the microcapsules from simulated gastrointestinal digestion (GID), where at the end of the intestinal phase, 82% of the initial phenolics were bioaccessible compared to 54% in the free GME. Besides, the encapsulated GME displayed a higher antioxidant activity by the ferric reducing antioxidant power assay than the free extract after GID. These results show the potential of this encapsulation system for applying GME as a nutraceutical with a high antioxidant capacity and protective effect against cellular oxidation.

Recent Advances in Cell-Based In Vitro Models to Recreate Human Intestinal Inflammation.

Inflammatory bowel disease causes a major burden to patients and healthcare systems, raising the need to develop effective therapies. Technological advances in cell culture, allied with ethical issues, have propelled in vitro models as essential tools to study disease aetiology, its progression, and possible therapies. Several cell-based in vitro models of intestinal inflammation have been used, varying in their complexity and methodology to induce inflammation. Immortalized cell lines are extensively used due to their long-term survival, in contrast to primary cultures that are short-lived but patient-specific. Recently, organoids and organ-chips have demonstrated great potential by being physiologically more relevant. This review aims to shed light on the intricate nature of intestinal inflammation and cover recent works that report cell-based in vitro models of human intestinal inflammation, encompassing diverse approaches and outcomes.

Microencapsulation of Gallic Acid Based on a Polymeric and pH-Sensitive Matrix of Pectin/Alginate.

The encapsulation of gallic acid (GA) through several methods has enhanced its shelf life and facilitated industrial applications. Polymeric matrices made of alginate and pectin were evaluated to encapsulate GA via spray drying. The pH-responsive release mechanism was monitored to validate the matrices' performances as wall materials and extend the bioactive compound stability. The microcapsules produced were characterized via scanning electron microscopy (SEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and cyclic voltammetry (CV). The retention and encapsulation efficiency ranges were 45-82% and 79-90%, respectively. The higher values were reached at 3 and 0.75% (w/v) pectin and sodium alginate, respectively. The scanning electron microscopy showed smooth spherical capsules and the average particle size ranged from 1327 to 1591 nm. Their performance and stability were evaluated with optimal results at a pH value of 7 throughout the investigation period. Therefore, this work demonstrated the suitability of gallic acid encapsulation via spray drying using pectin and alginate, which are biopolymers that can be obtained from circular economy processes starting from agro-industrial biomass. The developed formulations provide an alternative to protecting and controlling the release of GA, promoting its application in the food, pharmaceutical, and cosmetic industries and allowing for the release of compounds with high bioactive potential.

Encapsulation of vitamin D3 using rhamnolipids-based nanostructured lipid carriers.

This work had as main objective to encapsulate vitamin D3 (VD3) into nanostructured lipid carriers (NLCs) using rhamnolipids as surfactant. Glycerol monostearate and medium chain triglycerides with 2.625 % of VD3 were used as lipid materials. The three formulations of NLCs with VD3 (NLCs + VD3) were composed by 99 % of aqueous phase, 1 % of lipid phase and 0.05 % of surfactant. The difference between them was the ratio of solid:liquid in lipid phase. The NLCs + VD3 sizes ranged between 92.1 and 108.1 nm. The most stable formulation maintaining their caracteristics for 60 days at 4 °C. The NLCs + VD3 cytotoxicity demonstrated that concentrations of 0.25 mg/mL or lower up had a good biocompatibility in vitro. During the in vitro digestion, formulations with lower sizes and higher content on solid lipid had higher lipolysis rate and consequently higher VD3 bioaccessibility. The rhamnolipids-based NLCs are a good option for the encapsulation of VD3.

From mouth to gut: microfluidic in vitro simulation of human gastro-intestinal digestion and intestinal permeability.

Reproducible in vitro studies of bioaccessibility, intestinal absorption, and bioavailability are key to the successful development of novel food ingredients or drugs intended for oral administration. There is currently a lack of methods that offer the finesse required to study these parameters for valuable molecules typically found in small volumes - as is the case of nanomaterials, which are often used to carry and protect bioactives. Here, we describe a modular microfluidic-based platform for total simulation of the human gastro-intestinal tract. Digestion-chips and cell-based gut-chips were fabricated from PDMS by soft lithography. On-chip digestion was validated using a fluorescently labelled casein derivative, which followed typical Michaelis-Menten kinetics and showed temporal resolution and good agreement with well-established bench-top protocols. Irreversible inhibition of serine proteases using Pefabloc® SC and a 1 : 6 dilution was sufficient to mitigate the cytotoxicity of simulated digestion fluids. Caco-2/HT29-MTX co-cultures were grown on-chip under a continuous flow for 7 days to obtain a differentiated cell monolayer forming a 3D villi-like epithelium with clear tight junction formation, and with an apparent permeability (Papp) of Lucifer Yellow closely approximating values reported ex vivo (3.7 × 10-6 ± 1.4 × 10-6vs. 4.0 × 10-6 ± 2.2 × 10-6). Digesta from the digestion-chips were flowed through the gut-chip, demonstrating the capacity to study sample digestion and intestinal permeability in a single microfluidic platform holding great promise for use in pharmacokinetic studies.

Bacteriophage Delivery Systems for Food Applications: Opportunities and Perspectives.

Currently, one-third of all food produced worldwide is wasted or lost, and bacterial contamination is one of the main reasons. Moreover, foodborne diseases are a severe problem, causing more than 420,000 deaths and nearly 600 million illnesses yearly, demanding more attention to food safety. Thus, new solutions need to be explored to tackle these problems. A possible solution for bacterial contamination is using bacteriophages (phages), which are harmless to humans; these natural viruses can be used to prevent or reduce food contamination by foodborne pathogens. In this regard, several studies showed the effectiveness of phages against bacteria. However, when used in their free form, phages can lose infectivity, decreasing the application in foods. To overcome this problem, new delivery systems are being studied to incorporate phages and ensure prolonged activity and controlled release in food systems. This review focuses on the existent and new phage delivery systems applied in the food industry to promote food safety. Initially, an overview of phages, their main advantages, and challenges is presented, followed by the different delivery systems, focused in methodologies, and biomaterials that can be used. In the end, examples of phage applications in foods are disclosed and future perspectives are approached.

Edible alginate-based films with anti-SARS-CoV-2 activity.

The viability of SARS-CoV-2 on food surfaces and its propagation through the food chain has been discussed by several stakeholders, as it may represent a serious public health problem, bringing new challenges to the food system. This work shows for the first time that edible films can be used against SARS-CoV-2. Sodium alginate-based films containing gallic acid, geraniol, and green tea extract were evaluated in terms of their antiviral activity against SARS-CoV-2. The results showed that all these films have strong in vitro antiviral activity against this virus. However, a higher concentration of the active compound (1.25%) is needed for the film containing gallic acid to achieve similar results to those obtained for lower concentrations of geraniol and green tea extract (0.313%). Furthermore, critical concentrations of the active compounds in the films were used to evaluate their stability during storage. Results showed that gallic acid-loaded films lose their activity from the second week of storage, while films with geraniol and green tea extract only show a drop in activity after four weeks. These results highlight the possibility of using edible films and coatings as antiviral materials on food surfaces or food contact materials, which may help to reduce the spreading of viruses through the food chain.

Prospecting in silico antibacterial activity of a peptide from trypsin inhibitor isolated from tamarind seed.

Bacterial infections have become a global concern, stimulating the growing demand for natural and biologically safe therapeutic agents with antibacterial action. This study was evaluated the genotoxicity of the trypsin inhibitor isolated from tamarind seeds (TTI) and the antibacterial effect of TTI theoric model, number 56, and conformation number 287 (TTIp 56/287) and derived peptides in silico. TTI (0.3 and 0.6 mg.mL-1) did not cause genotoxicity in cells (p > 0.05). In silico, a greater interaction of TTIp 56/287 with the Gram-positive membrane (GP) was observed, with an interaction potential energy (IPE) of -1094.97 kcal.mol-1. In the TTIp 56/287-GP interaction, the Arginine, Threonine (Thr), and Lysine residues presented lower IPE. In molecular dynamics (MD), Peptidotrychyme59 (TVSQTPIDIPIGLPVR) showed an IPE of -518.08 kcal.mol-1 with the membrane of GP bacteria, and the Thr and Arginine residues showed the greater IPE. The results highlight new perspectives on TTI and its derived peptides antibacterial activity.

Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments.

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

Anti-Inflammatory Protein Isolated from Tamarind Promotes Better Histological Aspects in the Intestine Regardless of the Improvement of Intestinal Permeability in a Preclinical Study of Diet-Induced Obesity.

Obesity is associated with metabolic and physiological effects in the gut. In this study, we evaluated the anti-inflammatory effect of trypsin inhibitor isolated from tamarind seeds (TTI) in vitro (interaction with lipopolysaccharide (LPS) and inhibitory activity against human neutrophil elastase (HNE)), and using intestinal co-cultures of Caco-2:HT29-MTX cell lines inflamed with TNF-α (50 ng/mL) and a Wistar rat model of diet-induced obesity (n = 15). TTI was administered to animals by gavage (10 days), and the treated group (25 mg/kg/day) was compared to animals without treatment or treated with a nutritionally adequate diet. In the in vitro study, it showed inhibitory activity against HNE (93%). In co-cultures, there was no protection or recovery of the integrity of inflamed cell monolayers treated with TTI (1.0 mg/mL). In animals, TTI led to lower plasma concentrations of TNF-α and IL-6, total leukocytes, fasting glucose, and LDL-c (p < 0.05). The intestines demonstrated a lower degree of chronic enteritis, greater preservation of the submucosa, and greater intestinal wall thickness than the other groups (p = 0.042). Therefore, the better appearance of the intestine not reflected in the intestinal permeability added to the in vitro activity against HNE point to possibilities for new studies and applications related to this activity.

Histomorphometric analysis of the lung of Swiss mice treated with a fibrinolytic protease.

Fibrinolytic enzymes are considered promising alternative in the treatment of cardiovascular diseases by preventing fibrin clots. A protease from Mucor subtilissimus UCP 1262 was obtained by solid state fermentation and purified by ion exchange chromatography. The purified extract was administered at an acute dose of 2000 mg/mL to evaluate its toxic effects to the lungs of mice. After 14 days of treatment, a histomorphometric study was performed by the type 1 and 2 pneumocyte count and the evaluation of the lung area. As result, the experimental group showed a significant decrease of type 2 pneumocyte and although a decrease in the alveolar area was observed in relation to the control group, no significant pulmonary toxicity, emphysema, and fibrosis characteristics were detected. The in vitro tests suggest possible clinical applications for the enzyme.

Purification, biochemical characterization and fibrinolytic potential of proteases produced by bacteria of the genus Bacillus: a systematic literature review.

Thrombosis is a hematological disorder characterized by the formation of intravascular thrombi, which contributes to the development of cardiovascular diseases. Fibrinolytic enzymes are proteases that promote the hydrolysis of fibrin, promoting the dissolution of thrombi, contributing to the maintenance of adequate blood flow. The characterization of new effective, safe and low-cost fibrinolytic agents is an important strategy for the prevention and treatment of thrombosis. However, the development of new fibrinolytics requires the use of complex methodologies for purification, physicochemical characterization and evaluation of the action potential and toxicity of these enzymes. In this context, microbial enzymes produced by bacteria of the Bacillus genus are promising and widely researched sources to produce new fibrinolytics, with high thrombolytic potential and reduced toxicity. Thus, this review aims to provide a current and comprehensive understanding of the different Bacillus species used for the production of fibrinolytic proteases, highlighting the purification techniques, biochemical characteristics, enzymatic activity and toxicological evaluations used.

Zn and Zn-Fe Nanostructures with Multifunctional Properties as Components for Food Packaging Materials.

Metallic and bimetallic nanostructures have shown interesting chromatic and antibacterial properties, and they can be used in various applications. In this work, zinc (Zn) and iron (Fe) nanostructures were produced with different morphologies: (i) pure Zn; (ii) Zn-Fe nanoalloys; (iii) Zn-Fe nanolayers (Zn-Fe NLs); and (iv) Zn nanolayers combined with Fe nanoparticles (Zn NLs + Fe NPs). The aim was to produce components for food packaging materials with active and intelligent properties, including oxygen absorption capacity, chromatic properties, and antibacterial properties. Thus, the morphology, structure, and chemical composition of the samples were characterized and correlated with their oxidation, chromatic, and antibacterial properties. The results revealed a relevant reduction in the coating's opacity after oxidation varying from 100 to 10% depending on the morphology of the system. All coatings exhibited significant antibacterial activity against S. aureus, revealing a direct correlation with Zn content. The incorporation of Fe for all atomic arrangements showed a negative impact on the antibacterial effect against E. coli, decreasing to less than half the zone of inhibition for Zn-Fe NLs and Zn NLs + Fe NPs and suppressing the antibacterial effect for Zn-Fe alloy when compared with the pure Zn system.