Multiobjective optimization of frozen and freeze-dried Lactobacillus delbrueckii subsp. bulgaricus CFL1 production via the modification of fermentation conditions.

AIM: This study investigates the individual and combined effects of fermentation parameters for improving cell biomass productivity and the resistance to freezing, freeze-drying, and freeze-dried storage of Lactobacillus delbrueckii subsp. bulgaricus CFL1. METHODS AND RESULTS: Cells were cultivated at different temperatures (42°C and 37°C) and pH values (5.8 and 4.8) and harvested at various growth phases (mid-exponential, deceleration, and stationary growth phases). Specific acidifying activity was determined after fermentation, freezing, freeze-drying, and freeze-dried storage. Multiple regression analyses were performed to identify the effects of fermentation parameters on the specific acidifying activity losses and to generate the corresponding 3D response surfaces. A multiobjective decision approach was applied to optimize biomass productivity and specific acidifying activity. The temperature positively influenced biomass productivity, whereas low pH during growth reduced the loss of specific acidifying activity after freezing and freeze-drying. Furthermore, freeze-drying resistance was favored by increased harvest time. CONCLUSIONS: Productivity, and freezing and freeze-drying resistances of L. delbrueckii subsp. bulgaricus CFL1 were differentially affected by the fermentation parameters studied. There was no single fermentation condition that improved both productivity and resistance to freezing and freeze-drying. Thus, Pareto fronts were helpful to optimize productivity and resistance, when cells were grown at 42°C, pH 4.8, and harvested at the deceleration phase.

The complex puzzle of dietary silver nanoparticles, mucus and microbiota in the gut.

Hundreds of consumer and commercial products containing silver nanoparticles (AgNPs) are currently used in food, personal-care products, pharmaceutical, and many other applications. Human exposure to AgNPs includes oral intake, inhalation, and dermal contact. The aim of this review was to focus on oral intake, intentional and incidental of AgNPs where well-known antimicrobial characteristics that might affect the microbiome and mucus in the gastrointestinal tract (GIT). This critical review summarizes what is known regarding the impacts of AgNPs on gut homeostasis. It is fundamental to understand the forms of AgNPs and their physicochemical characterization before and during digestion. For example, lab-synthesized AgNPs differ from "real" ingestable AgNPs used as food additives and dietary supplements. Similarly, the gut environment alters the chemical and physical state of Ag that is ingested as AgNPs. Emerging research on in vitro and in vivo rodent and human indicated complex multi-directional relationships among AgNPs, the intestinal microbiota, and the epithelial mucus. It may be necessary to go beyond today's descriptive approach to a modeling-based ecosystem approach that might quantitatively integrate spatio-temporal interactions among microbial groups, host factors (e.g., mucus), and environmental factors, including lifestyle-based stressors. It is suggested that future research (1) utilize more representative AgNPs, focus on microbe/mucus interactions, (2) assess the effects of environmental stressors for longer and longitudinal conditions, and (3) be integrated using quantitative modeling.

The True Nature of Tricalcium Phosphate Used as Food Additive (E341(iii)).

Tricalcium phosphate (TCP) is a food additive, labeled E341(iii), used in powdered food preparation, such as baby formula. In the United States, calcium phosphate nano-objects were identified in baby formula extractions. Our goal is to determine whether the TCP food additive, as is used in Europe, can be classified as a nanomaterial. The physicochemical properties of TCP were characterized. Three different samples (from a chemical company and two manufacturers) were thoroughly characterized according to the recommendations of the European Food Safety Authority. A commercial TCP food additive was identified as actually being hydroxyapatite (HA). It presents itself in the form of particles of different shapes (either needle-like, rod, or pseudo-spherical), which were demonstrated in this paper to be of a nanometric dimension: E341(iii) is thus a nanomaterial. In water, HA particles sediment rapidly as agglomerates or aggregates over a pH of 6 and are progressively dissolved in acidic media (pH < 5) until the complete dissolution at a pH of 2. Consequently, since TCP may be considered as a nanomaterial on the European market, it raises the question of its potential persistency in the gastrointestinal tract.

Enhancement of the Anti-Angiogenic Effects of Delphinidin When Encapsulated within Small Extracellular Vesicles.

(1) Background: The anthocyanin delphinidin exhibits anti-angiogenic properties both in in vitro and in vivo angiogenesis models. However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (sEV) properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells (HAoECs). (2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide (NO) production and Matrigel assay were evaluated in HAoECs. (3) Results: Delphinidine, 3-O-ß-rutinoside and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs. sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation, 10-fold for endothelial NO production and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions: sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization.

In vitro digestion of food grade TiO2 (E171) and TiO2 nanoparticles: physicochemical characterization and impact on the activity of digestive enzymes.

Titanium dioxide is a food additive that has raised some concerns for humans due to the presence of nanoparticles. We were interested in knowing the fate of TiO2 particles in the gastro-intestinal tract and their potential effect on digestive enzymes. For this purpose, we analysed the behaviour of two different food grade TiO2 samples (E171) and one nano-sized TiO2 sample (P25) through a standardized static in vitro digestion protocol simulating the oral, gastric and intestinal phases with appropriate juices including enzymes. Both E171 and P25 TiO2 particles remained intact in the digestive fluids but formed large agglomerates, and especially in the intestinal fluid where up to 500 µm sized particles have been identified. The formation of these agglomerates is mediated by the adsorption of mainly α-amylase and divalent cations. Pepsin was also identified to adsorb onto TiO2 particles but only in the case of silica-covered E171. In the salivary conditions, TiO2 exerted an inhibitory action on the enzymatic activity of α-amylase. The activity was reduced by a factor dependent on enzyme concentrations (up to 34% at 1 mg mL-1) but this inhibitory effect was reduced to hardly 10% in the intestinal fluid. In the gastric phase, pepsin was not affected by any form of TiO2. Our results hint that food grade TiO2 has a limited impact on the global digestion of carbohydrates and proteins. However, the reduced activity specifically observed in the oral phase deserves deeper investigation to prevent any adverse health effects related to the slowdown of carbohydrate metabolism.

Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products.

Colloidal silver products are sold for a wide range of disinfectant and health applications. This has increased the potential for human exposure to silver nanoparticles (AgNPs) and ions (Ag+), for which oral ingestion is considered to be a major route of exposure. Our objective was to evaluate and compare the toxicity of two commercially available colloidal silver products on two human intestinal epithelial models under realistic exposure conditions. Mesosilver™ and AgC were characterized and a concentration range between 0.1 and 12 µg/mL chosen. Caco-2 cells vs. co-culture of Caco-2 and mucus-secreting HT29-MTX cells (90/10) were used. Repeated exposure was carried out to determine cell viability over 18 days of cell differentiation in 24-well plates. Selected concentrations (0.1, 1, and 3 µg/mL) were tested on cells cultured in E-plates and Transwells with the same repeated exposure regimen, to determine cell impedance, and cell viability and trans-epithelial electrical resistance (TEER), respectively. Silver uptake, intracellular localisation, and translocation were determined by CytoViva™, HIM-SIMS, and ICP-MS. Genotoxicity was determined on acutely-exposed proliferating Caco-2 cells by γH2AX immunofluorescence staining. Repeated exposure of a given concentration of AgC, which is composed solely of ionic silver, generally exerted more toxic effects on Caco-2 cells than Mesosilver™, which contains a mix of AgNPs and ionic silver. Due to its patchy structure, the presence of mucus in the Caco-2/HT29-MTX co-culture only slightly mitigated the deleterious effects on cell viability. Increased genotoxicity was observed for AgC on proliferating Caco-2 cells. Silver uptake, intracellular localisation, and translocation were similar. In conclusion, Mesosilver™ and AgC colloidal silver products show different levels of gut toxicity due to the forms of distinct silver (AgNPs and/or Ag+) contained within. This study highlights the applicability of high-resolution (chemical) imaging to detect and localize silver and provides insights into its uptake mechanisms, intracellular fate and cellular effects.

Pectin-lipid assembly at the air-water interface: effect of the pectin charge distribution.

Pectins are anionic polysaccharides that are sensitive to cations, a property that is widely used in food science. The interactions of a cationic lipid film (dimethyldioctadecylammonium bromide, DODAB) with a set of pectins bearing the same charge, which was either distributed randomly or pseudorandomly or blockwise, are investigated. The combination of Brewster angle microscopy BAM and infrared reflection-absorption spectroscopy IRRAS at the air-water interface reveals that pectin strongly binds to the cationic lipid film in forming a stacked layer underneath the lipid film. The detailed vibrational study of this stable mixed film indicates furthermore that pectin induces a disorder in the internal structure of the cationic film. The strong binding induces a splitting of the carboxylate stretching mode of pectin that is pressure and charge distribution dependent. The occurrence of an intermediate plateau below the collapse of the mixed film originates probably from a change in conformation of the pectin structure underneath the film.

Evaluation of the content of TiO2 nanoparticles in the coatings of chewing gums.

Titanium dioxide is a metal oxide used as a white pigment in many food categories, including confectionery. Due to differences in the mass fraction of nanoparticles contained in TiO2, the estimated intakes of TiO2 nanoparticles differ by a factor of 10 in the literature. To resolve this problem, a better estimation of the mass of nanoparticles present in food products is needed. In this study, we focused our efforts on chewing gum, which is one of the food products contributing most to the intake of TiO2. The coatings of four kinds of chewing gum, where the presence of TiO2 was confirmed by Raman spectroscopy, were extracted in aqueous phases. The extracts were analysed by transmission electron microscopy (TEM), X-ray diffraction, Fourier Transform Raman spectroscopy, and inductively coupled plasma atomic emission spectroscopy (ICP-AES) to establish their chemical composition, crystallinity and size distribution. The coatings of the four chewing gums differ chemically from each other, and more specifically the amount of TiO2 varies from one coating to another. TiO2 particles constitute the entire coating of some chewing gums, whereas for others, TiO2 particles are embedded in an organic matrix and/or mixed with minerals like calcium carbonate, talc, or magnesium silicate. We found 1.1 ± 0.3 to 17.3 ± 0.9 mg TiO2 particles per piece of chewing gum, with a mean diameter of 135 ± 42 nm. TiO2 nanoparticles account for 19 ± 4% of all particles, which represents a mass fraction of 4.2 ± 0.1% on average. The intake of nanoparticles is thus highly dependent on the kind of chewing gum, with an estimated range extending from 0.04 ± 0.01 to 0.81 ± 0.04 mg of nano-TiO2 per piece of chewing gum. These data should serve to refine the exposure scenario.

The miscibility of milk sphingomyelin and cholesterol is affected by temperature and surface pressure in mixed Langmuir monolayers.

The miscibility of milk sphingomyelin (milk-SM) and cholesterol was investigated in this study. The effect of different physical states of milk-SM on its interactions with cholesterol was determined by the recording of isotherms of compression of Langmuir films for temperatures above and below the gel to Lα phase transition of milk-SM (Tm∼34°C). For T=15°CTm, the milk-SM molecules were in a LE phase regardless of the surface pressure applied. A phase diagram pressure - milk-SM/cholesterol composition was established. This study demonstrated that both temperature and surface pressure affected the miscibility between the milk-SM and cholesterol. The strongest attractive forces (i.e. condensing effect) were identified for 30mol% cholesterol when the milk-SM was in the LE phase state.

Impact of food grade and nano-TiO2 particles on a human intestinal community.

Titanium dioxide (TiO2) nanoparticles (NPs) are used as an additive (E171 or INS171) in foods such as gum, candy and puddings. To address concerns about the potential hazardous effects of ingested NPs, the toxicity of these food-grade NPs was investigated with a defined model intestinal bacterial community. Each titania preparation (food-grade TiO2 formulations, E171-1 and E171-6a) was tested at concentrations equivalent to those found in the human intestine after sampling 1-2 pieces of gum or candy (100-250 ppm). At the low concentrations used, neither the TiO2 food additives nor control TiO2 NPs had an impact on gas production and only a minor effect on fatty acids profiles (C16:00, C18:00, 15:1 w5c, 18:1 w9c and 18:1 w9c, p < 0.05). DNA profiles and phylogenetic distributions confirmed limited effects on the bacterial community, with a modest decrease in the relative abundance of the dominant Bacteroides ovatus in favor of Clostridium cocleatum (-13% and +14% respectively, p < 0.05). Such minor shifts in the treated consortia suggest that food grade and nano-TiO2 particles do not have a major effect on human gut microbiota when tested in vitro at relevant low concentrations. However, the cumulative effects of chronic TiO2 NP ingestion remain to be tested.

Criteria to define a more relevant reference sample of titanium dioxide in the context of food: a multiscale approach.

Titanium dioxide (TiO2) is a transition metal oxide widely used as a white pigment in various applications, including food. Due to the classification of TiO2 nanoparticles by the International Agency for Research on Cancer as potentially harmful for humans by inhalation, the presence of nanoparticles in food products needed to be confirmed by a set of independent studies. Seven samples of food-grade TiO2 (E171) were extensively characterised for their size distribution, crystallinity and surface properties by the currently recommended methods. All investigated E171 samples contained a fraction of nanoparticles, however, below the threshold defining the labelling of nanomaterial. On the basis of these results and a statistical analysis, E171 food-grade TiO2 totally differs from the reference material P25, confirming the few published data on this kind of particle. Therefore, the reference material P25 does not appear to be the most suitable model to study the fate of food-grade TiO2 in the gastrointestinal tract. The criteria currently to obtain a representative food-grade sample of TiO2 are the following: (1) crystalline-phase anatase, (2) a powder with an isoelectric point very close to 4.1, (3) a fraction of nanoparticles comprised between 15% and 45%, and (4) a low specific surface area around 10 m2 g-1.

The benefits of liposomes for chilling canine sperm for 4 days at 4°C.

This study comprises 3 experiments exploring the possible benefits and mechanism of action of liposomes for chilling (4°C) canine sperm over a period of 4 days. In the first experiment, 20 ejaculates collected from 5 Beagle dogs were chilled in an extender containing 6% low density lipoproteins (LDL) (Control), or one of 7 extenders containing different concentrations (2, 4, 6, 8, 10, 15, 20%) of liposomes (LIPO). These ejaculates were chilled over 4 days and motility was assessed daily using a Hamilton Thorne analyzer (HTM-IVOS, 14.0). The 2% LIPO obtained the best results (p=0.038) after four days (72.55% motile spermatozoa and 31.4% progressive spermatozoa). In experiment 2, 10 ejaculates were collected from same 5 dogs and chilled in 6% LDL or 2% LIPO-based extenders. Sperm integrity characteristics were assessed prior to refrigeration and every 48h for four days (D0, D2, and D4). Acrosome integrity was assessed using the FITC-PSA test (Fluorescein IsoThiocyanate-Pisum Sativum Agglutinin), plasma membrane (PM) integrity using both the hypo-osmotic swelling test (HOSt) and SYBR14/Propidium Iodide test (SYBR14/PI), and DNA integrity using the Acridine-Orange test (AO). The 2% LIPO extender provided equivalent preservation of sperm integrity parameters to the reference extender (6% LDL). In experiment 3, a Langmuir-Blodgett trough was used to evaluate the mechanistic interactions between LDL, LIPO, prostatic fluid, and the canine spermatozoal membrane during chilling. Results indicate that LDL and LIPO interact differently with the biomimetic membrane. The most likely conclusion of these findings is that LDL and liposomes employ different protective mechanisms during the chilling (4°C) of canine spermatozoa.

Interactions between phospholipids and titanium dioxide particles.

A systematic study was carried out on monolayer films and lipid vesicles to elucidate the interactions between membrane lipids and commercial particles of titanium dioxide TiO2 (TiO2-P25). Pressure-area isotherms of lipids at various pH values were recorded on a Langmuir trough with or without TiO2-P25 and NaCl in the subphase. Electrophoretic mobilities of lipid vesicles and TiO2-P25 particles were measured to identify the pH range where attractive electrostatic interactions between lipids and TiO2-P25 could take place. The results show that (i) the surface of TiO2-P25 particles interacts only with some phospholipids, (ii) the driving forces are electrostatic and (iii) non-electrostatic interactions were also observed, depending on the molecular structure. More precisely, the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphate monosodium salt (DMPA), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-1-glycerol (DMPG) and 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (TMCL) interacted strongly with the TiO2-P25 surface through electrostatic interactions, providing they were oppositely charged, i.e. for pH between 2 and 6.6. For TMCL and DMPG, interactions with the surface of TiO2-P25 in non-favourable electrostatic conditions, suggested another kind of binding, probably through the hydroxyl groups of the terminal glycerol. Weaker attractive interactions were demonstrated for 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) and the synthetic lipid dihexadecyl phosphate (DHP). For DMPS, the carboxylate group is involved in the adsorption onto TiO2. The other membrane lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and sphingomyelin (SM) did not interact with TiO2-P25 regardless of pH.

Effect of lateral heterogeneity in mixed surfactant-stabilized interfaces on the oxidation of unsaturated lipids in oil-in-water emulsions.

The development of lipid oxidation in oil-in-water (O/W) emulsions is widely influenced by the properties of the interfacial layer, which separates the oil and water phases. In this work, the effect of the structure of the interface on the oxidative stability of surfactant stabilized O/W emulsions was investigated. Emulsions were prepared with either single Tween 20 or Tween 20/co-surfactant mixtures in limiting amounts. The co-surfactants, Span 20 and monolauroyl glycerol have the same hydrophobic tail as Tween 20 but differ by the size and composition of their polar headgroup. Metal-initiated lipid oxidation, monitored through the measurement of oxygen uptake, formation of conjugated dienes and volatile compounds, developed more rapidly in the emulsions stabilized by the surfactant mixture than in the single Tween 20-stabilized emulsion. The reconstitution of Tween 20/co-surfactant films at the air-water interface and their surface-pressure isotherms highlighted that, contrary to single Tween 20 molecules, Tween 20/co-surfactant mixtures exhibited an heterogeneous distribution within the interfacial layer, offering probably easier access of water-soluble pro-oxidants to the oil phase. These observations provide direct information about the link between the homogeneity of the interface layer and the oxidative stability of emulsions.

Modifications of interfacial proteins in oil-in-water emulsions prior to and during lipid oxidation.

Lipid oxidation is a major cause for the degradation of biological systems and foods, but the intricate relationship between lipid oxidation and protein modifications in these complex multiphase systems remains unclear. The objective of this work was to have a spatial and temporal insight of the modifications undergone by the interfacial or the unadsorbed proteins in oil-in-water emulsions during lipid oxidation. Tryptophan fluorescence and oxygen uptake were monitored simultaneously during incubation in different conditions of protein-stabilized oil-in-water emulsions. Kinetic parameters demonstrated that protein modifications, highlighted by decrease of protein fluorescence, occurred as an early event in the sequence of the reactions. They concerned more specifically the proteins adsorbed at the oil/water interface. The reactions led in a latter stage to protein aggregation, carbonylation, and loss of protein solubility.

Quantification of unadsorbed protein and surfactant emulsifiers in oil-in-water emulsions.

Unadsorbed emulsifiers affect the physical and chemical behaviour of oil-in-water (O/W) emulsions. A simple methodology to quantify unadsorbed emulsifiers in the aqueous phase of O/W emulsions has been developed. Emulsions were centrifuged and filtered to separate the aqueous phase from the oil droplets and the concentration of unadsorbed emulsifiers in the aqueous phase determined. The quantification of unadsorbed surfactants based on the direct transesterification of their fatty acids was validated for Tween 20, Tween 80, citric acid ester (Citrem), Span 20 and monolauroyl glycerol. To determine unadsorbed proteins, results obtained with Folin-Ciocalteu reagent or UV-spectrophotometry were compared on emulsions stabilized by ß-lactoglobulin (BLG), ß-casein (BCN) or bovine serum albumin (BSA). The first method gave more accurate results especially during aging of emulsions in oxidative conditions. The whole methodology was applied to emulsions stabilized with single or mixed emulsifiers. This approach enables optimization of emulsion formulations and could be useful to follow changes in the levels of unadsorbed emulsifiers during physical or chemical aging processes.

Contribution of the interfacial layer to the protection of emulsified lipids against oxidation.

The oxidative stability of oil-in-water (O/W) emulsions is highly dependent on the type of emulsifier. The purpose of this work was to investigate the specific role of the adsorbed emulsifiers on lipid oxidation of O/W emulsions. Emulsions of similar droplet size distribution stabilized by minimum amounts of proteins or surfactants were oxidized at 25 °C in the presence of equimolar iron-EDTA complex. The pH and the amount of emulsifier in the aqueous phase were also varied to investigate the role of the droplet charge and the emulsifier in the aqueous phase. Oxygen uptake, conjugated dienes (CD), and volatile compound formation demonstrated that the protein-stabilized interfaces are less efficient at protecting emulsified lipids against oxidation than surfactant-stabilized interfaces. The antioxidant effect of unadsorbed proteins was also confirmed.

Engineering of caseins and modulation of their structures and interactions.

Beta-casein (beta-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air-water interface are not well known. In order to study how the forced dimerisation influences functional properties of beta-CN, recombinant wild-type beta-CN was produced and distal cysteinylated forms of recombinant beta-CN were engineered. We show that 1) cysteinylated beta-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air-water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of beta-CN.

Caleosin of Arabidopsis thaliana : Effect of Calcium on Functional and Structural Properties.

A non-radioactive blot binding assay has proved the capacity of a purified recombinant form of Arabidopsis thaliana caleosin (AtClo1), a key protein of this plant oil body, to bind calcium. Calcium affected recombinant caleosin aggregation state, solubility, and electrophoretic mobility on SDS-PAGE. The effect of calcium on interfacial behavior of recombinant caleosin was studied at three interfaces: air/water (A/W), purified oil/water (O/W), and air/phosholipid/water (A/PLs/W). Recombinant caleosin was able to decrease interfacial tension (IFT) at A/W and O/W interfaces as a function of concentration and calcium, whereas no interaction was detected at the A/PLs/W interface. Effect of calcium was time dependent, and its amplitude strongly varied with the interface considered. Reconstituted oil bodies were used to prove the involvement of recombinant caleosin in their calcium-driven aggregation and coalescence. Calcium ions at concentration as low as 100 nM were able to strongly modify the shape and aggregation state of purified oil bodies, as well as their behavior within a monolayer, reflecting potentially profound changes in their structure and dynamic.