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.

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.

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.

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.

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.

Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles.

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

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.

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.

The Effect of Molecular Weight on the Antimicrobial Activity of Chitosan from Loligo opalescens for Food Packaging Applications.

The growing requirement for sustainable processes has boosted the development of biodegradable plastic-based materials incorporating bioactive compounds obtained from waste, adding value to these products. Chitosan (Ch) is a biopolymer that can be obtained by deacetylation of chitin (found abundantly in waste from the fishery industry) and has valuable properties such as biocompatibility, biodegradability, antimicrobial activity, and easy film-forming ability. This study aimed to produce and characterize poly(lactic acid) (PLA) surfaces coated with ß-chitosan and ß-chitooligosaccharides from a Loligo opalescens pen with different molecular weights for application in the food industry. The PLA films with native and depolymerized Ch were functionalized through plasma oxygen treatment followed by dip-coating, and their physicochemical properties were assessed by Fourier-transform infrared spectroscopy, X-ray diffraction, water contact angle, and scanning electron microscopy. Their antimicrobial properties were assessed against Escherichia coli and Pseudomonas putida, where Ch-based surfaces reduced the number of biofilm viable, viable but nonculturable, and culturable cells by up to 73%, 74%, and 87%, respectively, compared to PLA. Biofilm growth inhibition was confirmed by confocal laser scanning microscopy. Results suggest that Ch films of higher molecular weight had higher antibiofilm activity under the food storage conditions mimicked in this work, contributing simultaneously to the reuse of marine waste.

Microalgae as a Potential Functional Ingredient: Evaluation of the Phytochemical Profile, Antioxidant Activity and In-Vitro Enzymatic Inhibitory Effect of Different Species.

The functional food market has been in a state of constant expansion due to the increasing awareness of the impact of the diet on human health. In the search for new natural resources that could act as a functional ingredient for the food industry, microalgae represent a promising alternative, considering their high nutritional value and biosynthesis of numerous bioactive compounds with reported biological properties. In the present work, the phytochemical profile, antioxidant activity, and enzymatic inhibitory effect aiming at different metabolic disorders (Alzheimer's disease, Type 2 diabetes, and obesity) were evaluated for the species Porphyridium purpureum, Chlorella vulgaris, Arthorspira platensis, and Nannochloropsis oculata. All the species presented bioactive diversity and important antioxidant activity, demonstrating the potential to be used as functional ingredients. Particularly, P. purpureum and N. oculata exhibited higher carotenoid and polyphenol content, which was reflected in their superior biological effects. Moreover, the species P. purpureum exhibited remarkable enzymatic inhibition for all the analyses.

Development of Chitosan-Based Surfaces to Prevent Single- and Dual-Species Biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

Implantable medical devices (IMDs) are susceptible to microbial adhesion and biofilm formation, which lead to several clinical complications, including the occurrence of implant-associated infections. Polylactic acid (PLA) and its composites are currently used for the construction of IMDs. In addition, chitosan (CS) is a natural polymer that has been widely used in the medical field due to its antimicrobial and antibiofilm properties, which can be dependent on molecular weight (Mw). The present study aims to evaluate the performance of CS-based surfaces of different Mw to inhibit bacterial biofilm formation. For this purpose, CS-based surfaces were produced by dip-coating and the presence of CS and its derivatives onto PLA films, as well surface homogeneity were confirmed by contact angle measurements, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antimicrobial activity of the functionalized surfaces was evaluated against single- and dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. Chitosan-based surfaces were able to inhibit the development of single- and dual-species biofilms by reducing the number of total, viable, culturable, and viable but nonculturable cells up to 79%, 90%, 81%, and 96%, respectively, being their activity dependent on chitosan Mw. The effect of CS-based surfaces on the inhibition of biofilm formation was corroborated by biofilm structure analysis using confocal laser scanning microscopy (CLSM), which revealed a decrease in the biovolume and thickness of the biofilm formed on CS-based surfaces compared to PLA. Overall, these results support the potential of low Mw CS for coating polymeric devices such as IMDs where the two bacteria tested are common colonizers and reduce their biofilm formation.

Microalgae Encapsulation Systems for Food, Pharmaceutical and Cosmetics Applications.

Microalgae are microorganisms with a singular biochemical composition, including several biologically active compounds with proven pharmacological activities, such as anticancer, antioxidant and anti-inflammatory activities, among others. These properties make microalgae an interesting natural resource to be used as a functional ingredient, as well as in the prevention and treatment of diseases, or cosmetic formulations. Nevertheless, natural bioactives often possess inherent chemical instability and/or poor solubility, which are usually associated with low bioavailability. As such, their industrial potential as a health-promoting substance might be severely compromised. In this context, encapsulation systems are considered as a promising and emerging strategy to overcome these shortcomings due to the presence of a surrounding protective layer. Diverse systems have already been reported in the literature for natural bioactives, where some of them have been successfully applied to microalgae compounds. Therefore, this review focuses on exploring encapsulation systems for microalgae biomass, their extracts, or purified bioactives for food, pharmaceutical, and cosmetic purposes. Moreover, this work also covers the most common encapsulation techniques and types of coating materials used, along with the main findings regarding the beneficial effects of these systems.

Bio-Based Nanoparticles as a Carrier of ß-Carotene: Production, Characterisation and In Vitro Gastrointestinal Digestion.

ß-carotene loaded bio-based nanoparticles (NPs) were produced by the solvent-displacement method using two polymers: zein and ethylcellulose. The production of NPs was optimised through an experimental design and characterised in terms of average size and polydispersity index. The processing conditions that allowed to obtain NPs (<100 nm) were used for ß-carotene encapsulation. Then ß-carotene loaded NPs were characterised in terms of zeta potential and encapsulation efficiency. Transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction analysis were performed for further morphological and chemical characterisation. In the end, a static in vitro digestion following the INFOGEST protocol was performed and the bioaccessibility of ß-carotene encapsulated in both NPs was determined. Results show that the best conditions for a size-controlled production with a narrow size distribution are lower polymer concentrations and higher antisolvent concentrations. The encapsulation of ß-carotene in ethylcellulose NPs resulted in nanoparticles with a mean average size of 60 ± 9 nm and encapsulation efficiency of 74 ± 2%. ß-carotene loaded zein-based NPs resulted in a mean size of 83 ± 8 nm and encapsulation efficiency of 93 ± 4%. Results obtained from the in vitro digestion showed that ß-carotene bioaccessibility when encapsulated in zein NPs is 37 ± 1%, which is higher than the value of 8.3 ± 0.1% obtained for the ethylcellulose NPs.

Evaluation of linseed oil oleogels to partially replace pork backfat in fermented sausages.

BACKGROUND: Nowadays, fat replacement in meat products is a matter of concern in the meat industry. The objective of this study was to evaluate the replacement of pork backfat with two oleogels of linseed in dry-cured sausages. RESULTS: Five batches of dry-cured sausages were prepared with two oleogels, a mixture of γ-oryzanol and ß-sitosterol (SO) and beeswax (B), at two levels of replacement (20% and 40%) (SO-20, SO-40, B-20, and B-40, respectively) and a control batch. The fatty acid profile improved in terms of nutrition: the polyunsaturated fatty acid / saturated fatty acid (PUFA/SFA) and n-6/n-3 ratio was about 1.41 and 0.93 for the higher levels of replacement, SO-40 and B-40, respectively. Quality parameters such as pH and color also changed with the inclusion of oleogels, resulting in changes in the sensory quality. CONCLUSION: Oleogels based on linseed enabled the replacement of pork backfat in fermented sausages. Depending on the level of fat substitution, such oleogels could replace fat in dry-cured sausages at the industrial level. © 2019 Society of Chemical Industry.

Effect of acute exposure in swiss mice (Mus musculus) to a fibrinolytic protease produced by Mucor subtilissimus UCP 1262: An histomorphometric, genotoxic and cytological approach.

The fibrinolytic enzyme produced by Mucor subtilissimus UCP 1262 was obtained by solid fermentation and purified by ion exchange chromatography using DEAE-Sephadex A50. The enzyme toxicity was evaluated using mammalian cell lineages: HEK-293, J774.A1, Sarcoma-180 and PBMCs which appeared to be viable at a level of 80%. The biochemical parameters of the mice treated with an acute dose of enzyme (2000 mg/mL) identified alterations of AST and ALT and the histomorphometric analysis of the liver showed a loss of endothelial cells (P < 0.001). However, these changes are considered minimal to affirm that there was a significant degree of hepatotoxicity. The comet assay and the micronucleus test did not identify damage in the DNA of the erythrocytes of the animals treated. The protease did not degrade the Aα and Bß chains of human and bovine fibrinogens, thus indicating that it does not act as anticoagulant, but rather as a fibrinolytic agent. The assay performed to assess blood biocompatibility shows that at dose of 0.3-5 mg/mL the hemolytic grade is considered insignificant. Moreover, the enzyme did not prolong bleeding time in mice when dosed with 1 mg/kg. These results indicate that this enzyme produced is a potential competitor for developing novel antithrombotic drugs.

Sterol-based oleogels' characterization envisioning food applications.

BACKGROUND: Phytosterols, in particular a mixture of pure γ-oryzanol and ß-sitosterol, develop a tubular system that is able to structure oil. In this study, different concentrations of a combination of γ-oryzanol and a commercial phytosterol mixture, Vitaesterol®, were used for the development of edible oil oleogels. RESULTS: Small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD) were used to characterize at nano and molecular scale the aforementioned oleogels and confirm the formation of sterols-based hollow tubule structures. Increased hardness was observed with the increase of gelator content used in oleogel manufacturing. The produced oleogels showed promising features such as tailored mechanical strength and low opacity, which are important features when considering their incorporation into food products. CONCLUSION: Despite differences in gel strength, oleogels exhibited textural characteristics that make these structures suitable for incorporation in food products. The oil migration profile associated with these oleogels can provide a solution for the controlled release of lipophilic compounds as well as for the retention of oil in cooked food products. © 2018 Society of Chemical Industry.

Enzymatic production of xylooligosaccharides from Brazilian Syrah grape pomace flour: a green alternative to conventional methods for adding value to agricultural by- products.

BACKGROUND: The aim of this work was to determine the most favorable conditions for the production of xylooligosaccharides (XOS) from Brazilian Syrah grape pomace. Chemical processes were performed using a rotatable central composite design where the concentration of sulfuric acid or sodium hydroxide and the grape pomace flour/solvent mass ratio were the dependent variables. Enzymatic production was also evaluated using xylanase produced by Aspergillus niger 3T5B8 and Viscozyme® enzymatic commercial cocktail. RESULTS: Chemical extraction allowed to recover 21.8-74.6% and 5.2-96.3% of total XOS for acidic and alkaline processes respectively. Enzymatic production extracted up to 88.68 ± 0.12% of total XOS using xylanase and up to 84.09 ± 2.40% with Viscozyme® . CONCLUSION: The present study demonstrated different feasible methods to produce high-added-value molecules, i.e. XOS, from Syrah grape pomace flour, valorizing this major by-product. The use of enzymatic cocktails demonstrated to be an alternative to the conventional methods, allowing to obtain an eco-friendly and sustainable grape pomace extract. © 2018 Society of Chemical Industry.

Edible Films and Coatings as Carriers of Living Microorganisms: A New Strategy Towards Biopreservation and Healthier Foods.

Edible films and coatings have been extensively studied in recent years due to their unique properties and advantages over more traditional conservation techniques. Edible films and coatings improve shelf life and food quality, by providing a protective barrier against physical and mechanical damage, and by creating a controlled atmosphere and acting as a semipermeable barrier for gases, vapor, and water. Edible films and coatings are produced using naturally derived materials, such as polysaccharides, proteins, and lipids, or a mixture of these materials. These films and coatings also offer the possibility of incorporating different functional ingredients such as nutraceuticals, antioxidants, antimicrobials, flavoring, and coloring agents. Films and coatings are also able to incorporate living microorganisms. In the last decade, several works reported the incorporation of bacteria to confer probiotic or antimicrobial properties to these films and coatings. The incorporation of probiotic bacteria in films and coatings allows them to reach the consumers' gut in adequate amounts to confer health benefits to the host, thus creating an added value to the food product. Also, other microorganisms, either bacteria or yeast, can be incorporated into edible films in a biocontrol approach to extend the shelf life of food products. The incorporation of yeasts in films and coatings has been suggested primarily for the control of the postharvest disease. This work provides a comprehensive review of the use of edible films and coatings for the incorporation of living microorganisms, aiming at the biopreservation and probiotic ability of food products.

Design of whey protein nanostructures for incorporation and release of nutraceutical compounds in food.

Whey proteins are widely used as nutritional and functional ingredients in formulated foods because they are relatively inexpensive, generally recognized as safe (GRAS) ingredient, and possess important biological, physical, and chemical functionalities. Denaturation and aggregation behavior of these proteins is of particular relevance toward manufacture of novel nanostructures with a number of potential uses. When these processes are properly engineered and controlled, whey proteins may be formed into nanohydrogels, nanofibrils, or nanotubes and be used as carrier of bioactive compounds. This review intends to discuss the latest understandings of nanoscale phenomena of whey protein denaturation and aggregation that may contribute for the design of protein nanostructures. Whey protein aggregation and gelation pathways under different processing and environmental conditions such as microwave heating, high voltage, and moderate electrical fields, high pressure, temperature, pH, and ionic strength were critically assessed. Moreover, several potential applications of nanohydrogels, nanofibrils, and nanotubes for controlled release of nutraceutical compounds (e.g. probiotics, vitamins, antioxidants, and peptides) were also included. Controlling the size of protein networks at nanoscale through application of different processing and environmental conditions can open perspectives for development of nanostructures with new or improved functionalities for incorporation and release of nutraceuticals in food matrices.

Pediocin SA-1: A selective bacteriocin for controlling Listeria monocytogenes in maize silages.

In this study, we assessed the potential as silage additive of a bacteriocin produced by Pediococcus acidilactici Northern Regional Research Laboratory (NRRL) B-5627 (pediocin SA-1). Maize was inoculated either with a bacterial starter alone (I) or in combination with the bacteriocin (IP), and untreated silage served as control. We monitored the products of fermentation (ethanol, and lactic and acetic acids), the microbial population, and the presence of the indicator strain Listeria monocytogenes Colección Española de Cultivos Tipo (CECT) 4032 (1×10(5) cfu/g) after 1, 2, 5, 8, 16, and 30d of ensiling. Our results indicated antilisterial activity of the bacteriocin, anticipating the disappearance of L. monocytogenes in IP compared with I and control silages. The PCR-denaturing gradient gel electrophoresis analysis revealed the addition of the bacteriocin did not affect the bacterial communities of the spontaneous fermentation, and the inoculant-containing bacteria (Lactobacillus plantarum, Lactobacillus buchneri, and Enterococcus faecium) were found in addition to the bacterial communities of untreated maize silages in I and IP silages. Both treatments increased the concentration of antimicrobial compounds (acetic acid, ethanol, and 1,2-propanodiol) and led to lower residual sugar contents compared with the control, which would provide enhanced aerobic stability. The fact that the identified species L. plantarum, L. buchneri, and E. faecium produce some of these inhibitory compounds, together with their persistence throughout the 30d of fermentation, suggest these bacteria could actively participate in the ensiling process. According to these results, pediocin SA-1 could be used as an additive to control the presence of L. monocytogenes in maize silages selectively, while improving their fermentative quality and eventually their aerobic stability.